Rfl 576 


.6 



.N7 T69 
2002 
Copy 2 


FT MEPDE 
GenCol 1 















EPA/600/R-02/028 
December 2002 



Toxicological Effects of Fine Particulate 
Matter Derived from the Destruction of 

the World Trade Center 


National Health and Environmental Effects Research Laboratory 
Office of Research and Development 
U.S. Environmental Protection Agency 
Research Triangle Park, North Carolina 27711 


Notice 


(£/ 


This report has been reviewed and approved for release by the National Health and 
Environmental Effects Research Laboratory of the US Environmental Protection Agency. Approval 
does not signify that the contents necessarily reflect the views and policies of the Agency, nor does 
mention of trade names or commercial products constitute endorsement or recommendation for use. 
This report has been audited for quality assurance purposes and a Quality Assurance statement is 
included. Supporting documentation and raw data are available from Dr. Stephen H. Gavett, 
National Health and Environmental Effects Research Laboratory (MD-82), U.S. Environmental 
Protection Agency, Research Triangle Park, NC 27711 (telephone 1-919-541-2555, e-mail 
gavett.stephen@epa.gov). 





Cover photographs courtesy of the Federal Emergency Management Agency 


11 





Contents 


Authors, Contributors, and Reviewers.v 

Executive Summary. vi 

I. Introduction. 1 

II. Materials and Methods. 3 

A. WTC PM Sample Collection and Size Fractionation.3 

B. Extraction of PM from Teflon Filters.4 

C. Control PM Samples Used in WTC2001 Study.5 

D. Physical and chemical analysis of solid (bulk and filter) samples. 6 

1. Scanning electron microscopy / energy-dispersive x-ray (SEM/EDX) analysis. 6 

2. X-ray diffraction (XRD) analysis. 6 

3. X-ray fluorescence (XRF) analysis.7 

4. Carbon fraction analysis.7 

E. Chemical analysis of liquid extracts of bulk and filter samples.7 

1. pH.7 

2. Endotoxin.7 

3. Inductively coupled plasma - atomic emission spectrometry (ICP-AES) and - mass 

spectrometry (ICP-MS).7 

4. Ion chromatography (IC) of deionized water extracts. 8 

F. Experimental Animals and Weight Randomization. 8 

G. Toxicological Endpoints: Experimental Design. 8 

1. Experiment A.9 

2. Experiment B.9 

3. Experiment C.9 

H. Oropharyngeal Aspiration of PM Samples.9 

I. Nose-Only Inhalation Exposure.10 

J. Respiratory Responses Assessed by Whole Body Plethysmography.10 

1. Immediate Airway Responses to PM 2 5 Exposure.10 

2. Airway Responsiveness to Methacholine Aerosol.11 

K. Diffusing Capacity of the Lung for Carbon Monoxide. 11 

L. Bronchoalveolar Lavage (BAL). 11 

M. Histopathology .12 

1. Lung histopathology.;.12 

2. Nasal histopathology.12 

N. Statistical Analysis .12 


111 









































III. Results.14 

A. Chemical analysis of solid samples and liquid extracts.14 

1. Endotoxin and pH levels.14 

2. Elemental and Ion Analysis.14 

3. Carbon analysis.16 

4. Compound analysis by XRD.16 

5. SEM/EDX analysis.17 

6 . Summary.18 

B. Experiment A: Dose-Response Relationships of WTC PM 2 5 .19 

1. Body weights and immediate airway responses.19 

2. DECO.20 

3. BAL parameters.20 

4. Responsiveness to methacholine aerosol.22 

5. Lung histopathology.22 

6 . Summary.25 

C. Experiment B: Effects of Nose-Only Inhalation Exposure.25 

1. Exposure results.25 

2. Body weights.26 

3. Immediate airway responses to nose-only exposure.26 

4. DLCO measurements.27 

5. Responsiveness to methacholine aerosol.27 

6 . BAL parameters.29 

7. Nasal histopathology.30 

8 . Lung histopathology.31 

9. Summary.31 

D. Experiment C: Effect of Geographical Location of WTC PM Samples on Responses . 31 

1. Sub-experiments and body weights.31 

2. Responsiveness to methacholine aerosol.32 

3. BAL cells.35 

4. BAL proteins and enzymes.38 

5. Lung histopathology.38 

6 . Summary.41 

IV. Discussion.44 

V. Quality Assurance Statement.48 

VI. References.50 


IV 








































Authors, Contributors, and Reviewers 


Authors 

Stephen H. Gavett, Najwa Haykal-Coates, John K. McGee, Jerry W. Highfill, Allen D. Ledbetter, 
and Daniel L. Costa—National Health and Environmental Effects Research Laboratory (MD- 
82), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. 


Contributors 

John J, Vandenberg, Thomas J. Hughes, Brenda T. Culpepper, M. Ian Gilmour, Judy H. Richards, 
Paul A. Evansky, Dock Terrell, James R. Lehmann, Elizabeth H. Boykin, Mette J. Schladweiler, 
and Hassell G. Hilliard — National Health and Environmental Effects Research Laboratory, 
U.S. Environmental Protection Agency, Research Triangle Park, NC. 

Lung Chi Chen, Mitchell D. Cohen, Glenn R. Chee, Colette M. Prophete, and Jessica Duffy — New 
York University, Tuxedo, NY (supported by NIEHS Center grant ES00260 and EPA PM Center 
grant R827351). 

Glen E. Marrs and Staff — Experimental Pathology Laboratories, Research Triangle Park, NC. 

Jack R. Harkema and James G. Wagner — Michigan State University, East Lansing, MI. 

Shirley J. Wasson — National Risk Management Research Laboratory, U.S. Environmental 
Protection Agency, Research Triangle Park, NC. 

Teri L. Conner—National Exposure Research Laboratory, U.S. Environmental Protection Agency, 
Research Triangle Park, NC. 

Annette S. King and A. Glenn Ross — NCCBA / Senior Environmental Employment Program, 
Research Triangle Park, NC. 

Dennis D. Williams and William D. Ellenson — ManTech Environmental, Research Triangle Park, 
NC. 

Robert A. Cary and David F. Smith — Sunset Laboratory, Hillsborough, NC. 


Reviewers 

John B. Morris — University of Connecticut, Storrs, CT. 

Michelle M. Schaper — Mine Safety and Health Administration, Pittsburgh, PA. 

Michael C. Madden — National Health and Environmental Effects Research Laboratory, U.S. 
Environmental Protection Agency, Chapel Hill, NC. 


V 












Executive Summary 


The goal of the experiments described in this report was to evaluate the toxicity of fine 
particulate matter (PM) derived from the destruction of the World Trade Center (WTC) on the 
respiratory tract of mice, and thereby contribute to the short-term health risk assessment of WTC PM 
being conducted by the Environmental Protection Agency. The adopted approach allowed a 
comparison of the intrinsic acute toxicity of fine WTC PM in the respiratory tract to well-studied 
PM reference samples that range in toxicity from essentially inert to quite toxic. The fundamental 
question was whether fine WTC PM was uniquely highly toxic. 

This toxicological research complements efforts by EPA and other organizations to assess the 
extent and level of worker and public exposures to PM derived from the WTC disaster and recovery 
efforts. This research is informative, but it is of limited scope, with a focus on the toxicological 
effects of the fine fraction of WTC dust from a single exposure. A more complete characterization 
of potential health effects would include consideration of other size fractions, repeated exposures, 
additional doses and endpoints, and responses in species or strains of differing sensitivity. It was 
not possible to assess these other considerations in the present study. 

Fallen dust samples were collected on September 12 and 13 from various sites around Ground 
Zero, and the fine PM fraction (< 2.5 microns in diameter; PM, 5 ) was isolated on filters. PM 2 5 was 
extracted from the filters and extensively analyzed by several chemical and physical techniques. A 
dose-response study in mice was conducted comparing aspirated WTC PM 25 (pooled from 7 
different locations near the WTC site) with low and high toxicity PM 25 control samples (Mt. St. 
Helens and residual oil fly ash (ROFA), respectively). An acute nose-only inhalation exposure study 
was conducted on one WTC PM 25 sample, since upper airway irritation has been a primary 
complaint of those living and working in the WTC area. Finally, a short-term time course study was 
conducted comparing aspirated samples from the 7 different locations with each other and with a 
standard PM 2 5 sample (NIST 1649a, an ambient air PM sample collected in Washington, DC). 

Fine size-fractionated WTC PM 2 5 was composed primarily of calcium-based compounds such 
as calcium sulfate (gypsum) and calcium carbonate (calcite, the main component of limestone). 
These and other compounds and elements found in the WTC PM 2 5 samples are indicative of crushed 
building materials such as cement, concrete aggregate, ceiling tiles, and wallboard. Levels of carbon 
were relatively low, suggesting that combustion-derived particles did not form a significant fraction 
of these samples recovered in the immediate aftermath of the destruction of the towers. Gypsum and 
calcite are known to cause irritation of the mucus membranes of the eyes and respiratory tract. 

Samples of WTC PM 2 5 induced mild to moderate degrees of inflammation when administered 
at a relatively high dose (100 fig) directly into the airways of mice. The pulmonary inflammatory 
response was not as great as that caused by the reference PM 2 5 samples (toxic ROFA and ambient 
air NIST 1649a). However, this same dose of WTC PM 2 5 caused airway hyperresponsiveness (a 


VI 








greater sensitivity to agents which constrict breathing passages) comparable to NIST 1649a and to 
a greater degree than ROFA. Doses of 10 and 32 pg administered directly into the airways, or 
inhalation at 10 mg/m^ did not induce significant inflammation or hyperresponsiveness. The 
significant degree of airway hyperresponsiveness induced by the high dose of WTC PM 2 5 implies 
that components of the dust can promote mechanisms of airway obstruction. 

The results from these studies indicate that a high dose of WTC dust as PM 2 5 would be 
necessary to elicit effects in healthy people. Hypothetical calculations are presented indicating that 
a healthy worker at Ground Zero would have to inhale about 425 pg/m^ WTC PM 2 5 for 8 hours to 
achieve the same dose per tracheobronchial surface area as occurred with the high dose of WTC 
PM 2 5 used in the mouse studies. These high concentrations are conceivable in the aftermath of the 
collapse of the towers when rescue and salvage efforts were in effect. Therefore a healthy worker 
without respiratory protection could have inhaled enough WTC PM 25 to cause pulmonary 
inflammation, airway hyperresponsiveness, and manifestations of sensory irritation such as cough. 
Species differences in responses to inhalation of WTC PM 2 5 are unknown and were not considered 
in these calculations. Individuals who are especially sensitive to inhalation of dusts, such as 
asthmatics, may experience these effects at lower doses of inhaled WTC PM 2 5. These studies 
suggest that most healthy people would not respond to a single exposure to moderately high WTC 
PM 2 5 levels (about 130 pg/m^ or less for 8 hours) with any adverse respiratory responses. However, 
it should be emphasized that the effects of chronic (long-term) or repeated exposures to lower levels 
of WTC PM 2 5, or the persistence of any respiratory effects are unknown and were not components 
of this study. Although only fine PM 2 5 was tested in these experiments, its composition was similar 
to coarser PM, suggesting that biological responses to both size fractions within the respiratory 
system may be similar. The results of these studies will need to be placed within the context of an 
overall risk assessment for exposures to pollutants generated by the World Trade Center disaster. 


Vll 










1. Introduction 


The World Trade Center (WTC) disaster sparked 
enormous concern about the quality of the environment in 
the surrounding neighborhoods. One of the immediate 
concerns was the effect of dust from the collapse and 
burning of the towers on breathing, especially in more 
susceptible individuals. Dust infiltrated indoors into 
homes and apartments, in many cases up to several inches 
in depth. Fires at the WTC site continued for several 
months before finally being extinguished, and emitted 
significant quantities of particulate matter (PM). 
Recovery and reconstruction efforts have also contributed 
to emissions of fine (< 2.5 microns; PM 2 5), coarse (> 2.5 
and < 10 microns; PM 2 5 .]o), and larger (> 10 microns) size 
PM fractions. The dust particles from the WTC site 
appear to be quite alkaline in nature, probably due to 
partial dissolution of concrete, gypsum, and glass fiber 
particles (USGS, 2002). As people are trying to move 
back, decisions must be made about cleaning procedures 
since potential exposure issues are associated with 
redispersal and residual dust. 

Those moving back to their homes as well as those 
who work in the area have reported throat irritation, 
cough, and other indications of mucous tissue sensory 
irritation (New York Times, 2001; Washington Post, 
2002). Nose and throat irritation may be caused by 
particles which deposit in the nasal passages and upper 
airways and stimulate sensory nerve reflexes (Costa and 
Schelegle, 1999). Airborne dust may elicit inflammation, 
mucus production, coughing, and sneezing in an effort to 
clear the lung of particles (Raabe, 1999). However, 
inflammation, mucus production, and airway 
hyperresponsiveness may all contribute to airway 
obstruction. Since asthma is characterized by all of these 
cardinal features (Sears, 1997), it is logical to suspect that 
asthmatic individuals may be more sensitive to agents 
which further promote airway obstruction. 

The National Exposure Research Laboratory (NERL, 
USEPA), in coordination with Region 2 of the U.S. 
Environmental Protection Agency (USEPA or EPA) and 
the New York Department of Environmental Protection 
(NYDEP), has been monitoring ambient pollutants 


including volatile organic compounds (VOCs), dioxins, 
and PM in an effort to ascertain exposures. In addition. 
New York University (NYU) and Rutgers University have 
collected bulk samples of ash and dust in the immediate 
aftermath of the disaster. The National Health and 
Environmental Effects Research Laboratory (NHEERL, 
USEPA) has collaborated with these organizations to 
study health effects of PM from the immediate vicinity of 
the WTC site. 

The primary goal of the present study was to evaluate 
the potential health effects of PM in people working or 
living in the vicinity of the WTC and downwind of fires 
and dispersed building materials immediately after the 
WTC collapse. Toxicologic assessment of entrained 
(settled) dusts and combustion-derived PM dispersed in 
the areas surrounding the WTC will provide basic hazard 
identification information from which a broad health 
assessment may be derived. These findings would provide 
objective information to EPA, New York State, and local 
authorities to communicate to the public about collateral 
public health concerns. 

In order to begin assessment of the toxicity of dust 
derived from the destruction of the WTC towers, scientists 
from NYU (led by Drs. Lung Chi Chen and Mitchell 
Cohen) went to the area 2 iround “Ground Zero” on 
September 12 and September 13, 2001. They collected 
bulk samples of settled dust from several sites in the 
immediate vicinity (<0.5 miles). Back in their laboratories 
at NYU, they utilized a procedure to size-fractionate the 
dust to obtain both fine and coarse PM fractions which can 
be readily inhaled and deposit in the respiratory tract, and 
are therefore relevant for study of toxicological effects. 
On October 2, 2001, Dr. Chen contacted Dr. Daniel L. 
Costa of the U.S. EPA NHEERL in order to collaborate on 
investigations of the toxicity of these size-fractionated 
WTC PM samples. 

The approach of the present study (code name 
WT C2001) was to compare the toxicity of samples of size- 
fractionated WTC PM 25 with previously tested PM 
samples in mice. Mice offer a number of advantages for 
toxicity studies: 1 ) less sample is needed to assess toxicity; 









2 ) the biology of the mouse has been intensively studied in 
the scientific literature; 3) a wide array of mouse-specific 
analytical reagents is available; and 4) we have extensive 
experience in assessing physiological responses, 
inflammation, and respiratory tract injury in mice exposed 
to other samples of air pollutants. The WTC PM 25 
samples were thoroughly characterized by a number of 
chemical and physical techniques in order to compare the 
composition of the samples with other reference samples. 
A dose-response study in mice was conducted comparing 
aspirated WTC PM 2 5 (pooled from 7 different locations 
near the WTC site) with low and high toxicity PM 2 5 
control samples. An acute inhalation exposure study was 
conducted on one WTC PM 2 5 sample, since upper airways 
irritation is a primary complaint of those living and 
working in the WTC area. Finally, a short-term time 
course study was conducted comparing aspirated samples 
from 7 different locations with each other and with a 
standard PM 2 5 sample. 


Several methods were common to all three of these 
experiments to determine the toxicological effects of WTC 
PM 25 . The ability of these PM 2 5 samples to affect 
respiratory tract responsiveness to aerosolized 
methacholine was determined. Since this chemical 
triggers airway narrowing, the test is appropriate to 
determine sensitivity to agents which induce airway 
obstruction. Bronchoalveolar lavage is a common 
standard technique which quantifies numbers of 
inflammatory cells and levels of proteins and enzymes 
indicative of lung injury. Lung pathological effects were 
assessed in a semi-quantitative fashion in all studies, and 
pathological effects in the nasal region were determined in 
the inhalation study. Comparison of the toxicological 
effects of dust derived from the destruction of the WTC 
with PM 25 samples which have been extensively 
characterized in the literature will be clearly beneficial and 
relevant to the overall assessment of health consequences 
of environmental pollutants related to this disaster. 


2 









II. Materials and Methods 


A. WTC PM Sample Collection and Size 

Fractionation 

On 9/12/2001 and 9/13/2001, scientists from New 
York University went to the WTC area to collect bulk 
samples of fallen dust. Using a paper scoop, bulk samples 
were taken from various outdoor locations (e.g. car hood, 
window ledge, park bench) as well as one indoor location, 
all of which appeared undisturbed since the collapse of the 
towers, as judged by the presence of a smooth uniform 
layer of dust and the absence of indicators of recent human 
activity. Thirteen samples were collected and labeled with 
numbers (1 - 13) on 9/12/2001, and six samples were 
collected and labeled with letters (A - F) on 9/13/2001. 
Samples were stored in 75 ml or 250 ml polystyrene 
flasks at room temperature. All samples were collected 
before rain fell on 9/14/2001, which certainly altered 
chemical and physical characteristics of the dust. Samples 
were taken back to NYU for processing to isolate different 
size fractions. 

Bulk samples of dust were sieved with a 53 p mesh 
screen (USA Standard Testing Sieves, Fisher Scientific, 
Pittsburgh, PA) on a shaker (Portable Sieve Shaker, Tyler 
Industrial Products, Mentor, OH). The sieved material 
(PM53) was aerosolized through a 10 p cut inlet to remove 
particles in the 10-53 p range and isolate the PM,o 
fraction. The PM,o fraction then passed through a 2.5 p 
cyclone (made in house) to remove the PM 2 5.10 (coarse) 
fraction and isolate the PM 2 5 (fine) fraction. The PM 2 5 
fraction was collected on Teflon filters (Pall Gelman 
Sciences, Port Washington, NY - Zefluor Supported 
PTFE, 2 micron pore size, 47 mm, part # P5PJ047). While 
fractionating the PM samples, the filters became loaded 
and slowed airflow. Consequently, loaded filters were 
replaced with fresh filters periodically, and about 10 — 40 
filters were used to completely size-fractionate each WTC 
sample. Analysis of the weights found in the 4 size 
fractions showed that roughly half of the sample was in the 
PM 53 sieved fraction. Of the PM 53 fraction, about 80-89% 
was in the 10 - 53 p size range, which is too large to use in 
respiratory toxicology studies since only 45% of 10 p 


particles are even inhalable in small laboratory animals 
(Menache et al., 1995), and deposition of particles greater 
than 5 pm is minimal (Raabe et al., 1988). The amount of 
the 2.5 - 10 p fraction was very small (0.04 -1.14 % of the 
PM 53 fraction, except 3.23% in sample 13) and was 
therefore not feasible to study. The PM 25 fraction, 
however, was present in large enough amounts (2.29 - 4.06 
% of PM 53 fraction) to study for potential respiratory 
health effects, and is toxicologically relevant since it is 
associated with epidemiological findings of health effects 
in humans (Dockery et al., 1993). [The sum of the size 
fraction percentages does not total 100 % of the original 
PM 53 fraction because of loss of sample during 
fractionation steps.] After examination of the available 
inventory of filters and the locations where the samples 



Figure 1. WTC dust samples were collected by New York 
University (NYU) scientists from 13 sites on 9/12/2001 
(numbers) and from 6 sites on 9/13/2001 (letters). Collection 
sites are shown only for samples used in the WTC2001 study. 
Map provided by MapQuest.com, Inc. 


3 

















- ■ 




c-fc'*' 


«• 

--Si- 

##» H 

' 

5 

A 

7 ®* 



Figure 2. WTC bulk dust samples were size-fractionated by 
NYU. Filters containing the PMj 5 fraction were received at the 
U.S. EPA in Research Triangle Park, NC on 10/26/2001, and 
were inspected and photographed 10/29/2001. 


information about the toxicity and mode of action of WTC 
PM 25 . Since there was not enough PM 25 or PM 25.10 
sample available to conduct an inhalation exposure study 
(> 2 g necessary), it was decided to use a PM 53 sample 
(sieved but not further fractionated) which was available 
in large enough quantities to run through the inhalation 
exposure system. The EPA inhalation exposure system 
has a 2.5 pm cut-point cyclone to remove larger particles 
(Ledbetter et al., 1998), and therefore measurement of the 
PM concentration in the exposure zone of the chamber 
represents exposure to PM2 5. A sample of PM53 from 
location #3 (figure 1), 0.3 miles east of Ground Zero (in 
the predominant wind direction), was available in large 
enough quantities for the nose-only inhalation exposure 
study. This sample was sent by overnight express from 
NYU and received on November 21, 2001. 


were collected, filters containing the PM 2 5 fraction were 
selected from seven locations (sites 8 , 11, 13, B, C, E, and 
F) around Ground Zero, in order to assess toxicity of 
samples from different geographical locations as well as 
overall toxicity of a pooled sample from these locations 
(Figure 1). The locations were selected to represent a 
distribution surrounding the WTC site, with more 
collection sites in the east reflecting the predominant 
winds in that direction. 

Fourteen Teflon filters containing the PM 2 5 fraction 
from the 7 different sites collected around the World 
Trade Center on 9/12/01 and 9/13/01 were shipped by 
overnight express to EPA and received on October 26, 
2001 , and these were inspected and photographed on 
October 29, 2001 (Figure 2). [Throughout the WTC2001 
study, sample transfers were accompanied by signed 
chain-of-custody letters]. A total quantity of about 50 mg 
from each site, collected on 1 to 3 filters per site, was 
provided. The weight of PM 25 on the filters was 
determined by NYU, and was separately determined at 
EPA after overnight dessication using a Cahn 
electrobalance. The description of the locations of the 7 
samples and the total weight of PM 2 5 on the filters from 
each site is provided in Table 1. PM 25 could not be 
efficiently scraped off of one filter, so it was necessary to 
isolate the PM 2 5 using an aqueous extraction procedure 
(see below). 

Throat irritation, cough, nosebleeds, and other mucous 
tissue/sensory irritation were reported by residents and 
workers in the WTC area (Washington Post, 2002). 
Oropharyngeal aspiration of PM bypasses the nose and 
therefore potentially relevant effects may go undetected. 
Consequently, it was decided that an inhalation exposure 
study should be conducted which might reveal important 


B. Extraction of PM from Teflon Filters 

Filters were extracted using a modification of a 
method by Biran and coworkers (1996). Each filter was 
handled with clean sterilized stainless steel forceps. 
Filters from each of the 7 individual collection sites (1-3 
filters per site) were extracted into a single volume of 
distilled water (Gibco BRJL ultrapure 10977-015, lot 
1063705) in the ratio of 0.5 ml water per mg sample (2 mg 
PM / ml water; range 24.96 - 27.14 ml). This volume of 
water was pipetted into a 100 ml sterile plastic specimen 
cup containing a 3 mm thick Teflon ring at the bottom of 
the cup designed to support the filter. Filters were wetted 
with 200 pi of 70% ethanol on the particle side. The 
liquid was gently spread on the filter with the pipet tip, 
taking care not to scrape the filter. The filter was then 
placed on top of the 3 mm thick Teflon ring in the 
specimen cup with the particle side down, and a 6 mm 
thick Teflon ring was placed on top of the filter. The cup 
with the filter was secured to an orbital shaker (Titer Plate 
Shaker, Lab-Line Instruments, Melrose Park, IL). A 
cleaned sonicator probe (18 mm diameter. Sonic 300 
Dismembrator, Artek Systems Corp., Farmingdale, NY) 
was rinsed with 1% Triton X-100 (Sigma Chemical Co., 
St. Louis MO; T8787), and then ultrapure distilled water 
(Gibco BRL ultrapure 10977-015, lot 1063705) before and 
after each extraction. The probe was then lowered into the 
water in the specimen cup to a level just above the filter. 
Ice was placed around the specimen cup to prevent rising 
temperatures during sonication, and the temperature of the 
water was measured before and after sonication. The 
shaker was turned to the lowest speed at which it would 
run continuously (setting = 2). The sonicator power was 
set to 30, and the filter was sonicated for 30 minutes while 
rotating on the orbital shaker. After sonication, the filter 


4 










Table 1. Description of Samples Used in WTC2001 Study 


WTC Site Samples 


Experiment 

Sample 

Code 

Collection 

Date 

Location, Description 

Size 

Fraction 

Total Weight 
on Filters 

Extracted Wt., 

% Extracted 

A, C 

WTC 8 

9/12/2001 

Beekman Street - filters # 9, #14 

0.4 miles E of Ground 0 center 

PM,.3 

53.316 mg 

46.70 mg 
87.6% 

A,C 

WTC 11 

9/12/2001 

55 Church Street - filters #13, #14, #15 

In front of Millenium Hilton Hotel 

0.1 miles E of Ground 0 center 

PM2.5 

50.097 mg 

29.79 mg 
59.5% 

A, C 

WTC 13 

9/12/2001 

Church & Liberty St. - filters #4, #5 

0.1 miles SE of Ground 0 center 

PM3.S 

51.006 mg 

46.29 mg 
90.8% 

A,C 

WTC B 

9/13/2001 

Trinity & Rector - filter #4 

From a car hood and windshield 

0.25 miles S of Ground 0 center 

PM,.5 

52.969 mg 

42.31 mg 
79.9% 

A, C 

WTCC 

9/13/2001 

Winter Garden Park - filters #4, #7 

From a park bench facing the Hudson 

0.2 miles WNW of Ground 0 center 

PM2.5 

54.285 mg 

47.67 mg 
87.8% 

A, C 

WTC E 

9/13/2001 

Murray & Greenwich - filters #5, #6 

From a window ledge 

0.25 miles NNE of Ground 0 center 

PM2.5 

49.919 mg 

45.13 mg 
90.4% 

A,C 

WTC F 

9/13/2001 

Inside 120 Broadway - filters #2, #12 

From a marble staircase with no footprints 
0.25 miles SE of Ground 0 center 

PM,.5 

53.600 mg 

38.73 mg 
72.3% 

B 

WTC 3 

9/12/2001 

23 Park Row - Ground sample in front of 
J&R Electronics (across City Hall Park) 

0.3 miles E of Ground 0 center 

PM <53 

21.521 g 
sieved material 

sieved - not 
further 
fractionated 


Control PM Samples 


Experiment 

Sample 

Code 

Collection 

Date 

Description 

Size 

Fraction 

Weight 

Available 

Extracted Wt., 

% Extracted 

C 

NIST 

1976-1977 

NIST Standard Reference Material 1649a 
(Urban Dust collected in Washington DC) 

PM ,.5 

47.984 mg 

39.33 mg 
82.0% 

A 

MSH 

1980 

Mt. St. Helens ash, Washington State, from 
Graham et al., 1985 

PM3.5 

> lOg 

previously size- 
separated 

A 

ROFA 

1994 

Residual oil fly ash, ROFA Sample 3 from 
Kodavanti et al., 1998 

MMAD: 

2.665 

>2g 

milled - not 
extracted 


was gently removed with forceps and excess liquid was 
drained from the filter into the cup. Filters were placed 
back in their petri dishes, allowed to dry, and were 
dessicated before reweighing to determine quantity 
extracted (i.e. removed) from the filters (Table 1), The 
suspension of PM was thoroughly mixed, the pH was 
determined, and 10 ml was pipetted from each of the 7 
samples into a single sterilized 150 ml Erlenmeyer flask on 
ice to make a pooled sample (WTCX). The pH of the 
pooled sample was also determined. Of the remaining 
amount from each individual sample, 1 ml was taken for 
endotoxin analysis, and the remainder was pipetted into 
sterile 15 ml polystyrene tubes. 

The flask containing the pooled sample was covered 
with parafilm, and the pooled and individual site samples 


were frozen at -80 °C prior to lyophilization. Holes were 
poked in the parafilm of the pooled sample, while the caps 
on the 15 ml individual site sample tubes were loosened. 
Samples were lyophilized for 2 days at -55 °C and 140 
mtorr (Virtis Company, Gardiner, NY). After 
lyophilization, samples were stored at 4 °C until 
resuspension in sterile saline on the day of use in 
oropharyngeal aspiration. 

C. Control PM Samples Used in WTC2001 Study 

In order to assess toxicity of WTC PM 2 5 , pooled and 
individual site samples were compared with three other 
well-characterized PM 25 samples. Standard Reference 
Materials (SRM) are extensively characterized samples 
available from the National Institute of Standards and 


5 




















Technology (NIST, Gaithersburg, MD). SRM 1649a is an 
urban particulate matter sample which was collected in the 
Washington DC area in 1976-1977 over a 12 month period 
and represents a time-integrated sample (NIST, 2001). 
This material was selected in order to compare toxicity of 
WTC PM 2 5 with other typical urban air PM 2 5 (albeit from 
an earlier era when leaded gasoline was still in use). Since 
this material was collected as a total suspended particulate 
(TSP) sample with a large amount of coarse non-respirable 
PM, it was necessary to size-fractionate it in order to 
compare it with the WTC PM 2 5 samples. Vials of NIST 
1649a were purchased and then sent to NYU for size- 
fractionation using the same procedures as outlined above. 
The PM 2 5 fraction was sent back to EPA, and NIST 1649a 
was extracted from Teflon filters as described above. 

The toxicity of WTC PM 2 5 was also compared to that 
of a PM 2 5 fraction of ash from Mt. St. Helens (MSH) in 
Washington state (Graham et al., 1985). Approximately 
half of MSH is crystalline in nature, primarily plagioclase, 
a series of compounds beginning with NaAlSijOg and 
ending with CaAl 2 Si 208 which show continuous solid 
solution from albite to anorthite, with CaAl replacingNaSi 
as the series progresses. The remaining portion of MSH 
is amorphous (glass), while there are minor amounts of 
cristobalite (3%) and quartz (< 1%). The PM 2 5 fraction of 
MSH has low toxicity in rats (Raub et al., 1985) and mice 
(Hatch et al., 1984). Since the MSH sample had already 
been size-fractionated (Graham et al., 1985), it was not 
necessary for NYU to further size-fractionate it with their 
system. 

Residual oil fly ash (ROFA) is a fugitive fine PM 
sample with a high content of bioavailable transition 
metals including vanadium, nickel, and iron. Numerous 
studies by investigators at EPA and other institutions have 
demonstrated that these metals are associated with lung 
injury in both healthy animals and animal models of 
cardiopulmonary injury (Dreher et al., 1997; Gavett et al., 
1999, Kodavanti et al., 1998, Watkinson et al., 1998). For 
the WTC2001 study, we chose a sample of ROFA from a 
boiler system which is toxic yet not as soluble in water as 
previous samples of ROFA (ROFA sample #3 from 
Kodavanti et al., 1998), and is therefore more comparable 
to WTC PM samples which are not extremely water- 
soluble. The ROFA samples in the study by Kodavanti 
(1998) were reduced in size by placing each sample with 
a stainless-steel ball in a stainless-steel cup and shaking 
vigorously in a ball mill shaker for 30 - 60 minutes, and 
then passing the sample through a 100 p mesh nylon 
screen. ROFA sample #3 has a mass median aerodynamic 
diameter (MMAD) of 2.665 p. Although it was slightly 
larger in size compared with the other samples used in the 


WTC2001 study, it was decided that further size 
fractionation at NYU was not necessary. Control PM 
samples were stored at room temperature in polystyrene or 
polypropylene tubes shielded from light. See Table 1 for 
the summary descriptions of control PM samples. 

Samples of WTC PM, NIST, MSH, and ROFA were 
characterized by scanning electron microscopy / energy 
dispersive X-ray (SEM/EDX), X-ray diffraction (XRF), X- 
ray fluorescence (XRD), carbon fraction analysis, pH and 
endotoxin analysis, inductively coupled plasma-mass 
spectrometry / atomic emission spectrometry (ICP-MS / 
ICP-AES), and ion chromatography (IC). 

D. Physical and chemical analysis of solid (bulk and 

filter) samples. 

1. Scanning electron microscopy /energy-dispersive 
x-ray (SEM/EDX) analysis. SEM/EDX was used to 
obtain physical and chemical characteristics of particles 
and fibers found in bulk WTC2001 and control dust PM 2 5 
samples, and on polycarbonate filters taken during an 
inhalation exposure using the WTC3 sample. The 
Personal SEM® (PSEM) (formerly R. J. Lee Instruments, 
Ltd., now Aspex Instruments, Trafford, PA) was used to 
conduct the manual, single-particle analyses. The PSEM 
is a digital SEM/EDX system equipped with secondary 
and backscattered electron detectors for imaging, and a 
thin-window EDX detector enabling X-ray detection of 
carbon and heavier elements. For bulk samples, a small 
amount was applied to an adhesive carbon tab affixed to 
an aluminum SEM stub. For filter samples, small pieces 
(less than 1 cm^) were affixed to aluminum SEM stubs 
using a carbonaceous suspension. Images were created 
using the backscattered electron mode, which enhances the 
contrast of metals and other heavy elements with the 
background carbonaceous medium compared with lighter 
element particles. Photomicrographs of the individual 
features provide particle morphology and approximate 
physical size; an x-ray spectrum displayed below the 
image provides information on the elemental composition 
of the feature. SEM/EDX analysis was performed by the 
National Exposure Research Laboratory, Research 
Triangle Park, NC. Since only 15-30 images were 
examined from each sample, the results should not be 
interpreted as quantitative or comprehensive (Mamane et 
al., 2001). Rather, these qualitative results were primarily 
used to determine consistency with other analytical 
techniques described below. 

2. X-ray diffraction (XRD) analysis. XRD was used 
to determine qualitatively whether any crystalline 
compounds were present in sufficient quantity to be 
identified in the WTC3 sample used in the inhalation 


6 






exposure study. The bulk solid PM 53 sample was side- 
drifted into an aluminum holder and mounted into the 
Siemens D-500 Diffractometer (Bruker Analytical X-Ray 
Systems, Madison, WI). The generator, set at 45 kilovolts 
(kV) and 40 milliamperes (mA), generated x-rays from a 
copper-target x-ray tube. The filament current was 3.46 
mA. Intensities were collected by a lithium-drifted silicon 
detector fitted with a monochromator riding on a 
goniometer in the coupled 0/20 mode. Peaks were 
collected in the range 20 = 5 to 85 degrees. Collection 
software used was Materials Data, Inc. (MDI, Livermore, 
CA) Datascan, Version 3.2. Evaluation software was MDI 
Jade 5 using the pattern library Powder Diffraction File 
(PDF), release 2000 (International Centre for Diffraction 
Data). XRD analysis was performed by the National Risk 
Management Research Laboratory, Research Triangle 
Park, NC. 

3. X-ray fluorescence (XRF) analysis. Five 
polycarbonate filters (Isopore 0.8um #ATTP04700, 
Millipore Corporation, Bedford, MA) loaded with the 
WTC3 PM 2 5 sample used in the inhalation exposure study, 
along with five lot-matched blank filters, were loaded into 
liquid-type polyethylene sample cups, placed in a stainless 
steel sample holder, and analyzed. No film was used to 
cover blanks or samples in the analysis. X-ray intensities 
were collected with the Philips PW2404 XRF (Philips 
Analytical, Inc., Natick, MA), and the loaded particulate 
was analyzed using the “standardless” software, UniQuant 
4 , after subtraction of the counts due to the blank filter 
system (includes filter, polyethylene liquid sample cup and 
stainless steel sample holder). The intensities were 
averaged in each channel needed for background 
subtraction. The blank filter analysis showed slightly 
elevated counts due to Fe, Cr, Cu, Ca, Cl, S, and Si. The 
constituents of the dust were evaluated as oxides, but are 
reported quantitatively as elements with the oxygen 
stripped. XRF analysis was performed by the National 
Risk Management Research Laboratory, Research 
Triangle Park, NC. 

4. Carbon fraction analysis. Carbon fraction analysis 
was used to speciate the carbon content of samples into 
organic, elemental, and carbonate carbon. Analysis was 
performed on bulk WTC2001 and control dust PM 2 5 
samples, and on quartz filters taken during an inhalation 
exposure using WTC3 PM 2 5. The thermo-optical method, 
based upon sequential pyrolytic vaporization and detection 
of the three carbon fractions (Birch and Cary, 1996; 
Sunset, 2002), was performed by Sunset Laboratory, 
Forest Grove, OR (bulk samples), and Hillsborough, NC 
(filter samples). 


E. Chemical analysis of liquid extracts of bulk and 

filter samples. 

1. pH. The pH of samples isolated by aqueous 
extraction was determined immediately after the extraction 
procedure with an audited calibrated Coming 440 pH 
meter (audited by Research Triangle Institute, Research 
Triangle Park, NC). 

2. Endotoxin. Aliquots of samples isolated by 
aqueous extraction were frozen on dry ice and sent by 
overnight delivery to Associates of Cape Cod, Inc. 
(Falmouth, MA) for analysis of endotoxin content using 
the Limulus Amebocyte Lysate (LAL) gel-clot method. 
LAL-reagent water (lot # 308-331) was used to 
reconstitute or dilute Pyrotell lysate, endotoxin, and 
samples, and served as the negative control. Samples were 
titered using a twofold dilution scheme against control 
standard endotoxin (CSE; lot #85, Escherichia coli 0113, 
5 EU/ng). Preliminary inhibition tests (positive product 
controls) were performed on the undiluted samples spiked 
with CSE equivalent to twice the sensitivity {X\ 0.03 
EU/ml). The error of the gel-clot method is ± one twofold 
dilution. 

3. Inductively coupled plasma - atomic emission 
spectrometry (ICP-AES) and-mass spectrometry (ICP- 
MS). WTC2001 PM 2 5 samples, control dust PM 2.5 
samples, and polycarbonate filters taken during an 
inhalation exposure using WTC3 PM 2 5 were extracted 
with deionized (d.i.) water or IM HCl, and analyzed for 
their elemental content. The two extraction liquids are 
used to estimate easily bioavailable and total bioavailable 
metal content, respectively. While this speciation scheme 
is a rough approximation of bioavailability, it has proved 
useful in characterizing inhalation toxicology endpoints 
for various source and ambient particulates (Costa and 
Dreher, 1997; Kodavanti et al., 1998). Milligram-sized 
aliquots of bulk samples were extracted with 1.6 ml of 
either liquid. Polycarbonate filters were extracted with 13 
ml of either liquid. High-speed centrifugation was used to 
separate the liquid and solids (17000 x g for 1.6 ml 
samples, 51000 x g for 13 ml samples). After dilution, 
extraction solutions were analyzed quantitatively using 
ICP-AES (Model P40, PerkinElmer Instruments, Shelton, 
CT) operated closely following EPA Method 200.7 (EPA, 
2002a), and ICP-MS (ELAN 6000, PerkinElmer 
Instruments, Shelton, CT) operated closely following EPA 
Method 6020 (EPA, 2002b). Blank Gelman Teflo and 
Millipore Isopore filters (used in the inhalation study) 
were run through the extraction procedure. Filter blanks 
levels for all elements were negligible compared to the 
levels in the PM samples. Gelman Zefluor and Teflo 
filters and Millipore Isopore filters are of known, similar 


7 







low background levels. These filters are all produced for 
air particulate sampling and are commonly used for 
chemical analysis since their background chemical levels 
are negligible relative to the mass of samples amounts in 
this study. ICP-AES and ICP-MS analyses were 
performed by the National Health and Environmental 
Effects Laboratory, Research Triangle Park, NC. 

4. Ion chromatography (IC) of deionized water 
extracts. Deionized water extracts from the ICP sample 
prep as described above were analyzed quantitatively for 
anion and cation content using IC (DX-500, Dionex, 
Sunnywale, CA). The ASM column was used for anion 
analysis and the CS12 column was used for cation 
analysis. IC analysis was performed by ManTech 
Environmental, an onsite contractor for the National 
Exposure Research Laboratory, Research Triangle Park, 
NC. 

F. Experimental Animals and Weight Randomization. 

Young adult (7 week old) female CD-I mice (an 
outbred strain) were obtained from Charles River Breeding 
Laboratory (Crl:CD-l® (ICR) BR) in Raleigh, NC or 
Portage, Ml (the latter used in Experiment A5 only). An 
outbred strain was chosen because results from any 
specific inbred strain might be applicable only to that 
strain. CD-I mice were selected since researchers in the 
Experimental Toxicology Division of the U.S. EPA have 
extensive experience with this strain, while females were 
chosen for convenience so that they could be housed 
together in groups corresponding to treatment. The health 
screening report of mice from the colony accompanied 
each shipment of animals and was evaluated to determine 
if there were pathogens detected in the colony which could 
potentially affect responses. In all shipments, no 
pathogens were detected which could affect respiratory 
responses. Mice were housed in plastic cages on beta-chip 
bedding in groups of 4 per cage in room JJ-4 of the animal 
colony of the Environmental Research Center, Research 
Triangle Park, NC. Food (Prolab RMH 3000) and water 
were provide ad libitum and cages were changed at least 
twice a week. Mice were maintained on a 12 hr light/dark 
cycle at approximately 22 °C and 50% relative humidity in 
our AAALAC-approved facility, and held for a minimum 
of 5 days before treatment. Monthly sentinel screens were 
negative for sendai, mouse hepatitis virus, mycoplasma 
pulmonis, CARbacillus, parvovirus, endo- and ecto¬ 
parasites, and pinworms. Protocols used in this study were 
reviewed and approved by the EPA Institutional Animal 
Care and Use Committee (Laboratory Animal Project 
Review number 02-03-003 with amendments), and were 
conducted using national guidelines for the care and 


protection of animals. 

In all experiments, mice were randomly assigned to 
exposure groups based on weights. The weight 
randomization program (RandomVB) was developed in- 
house, validated, and documented in operating procedure 
OP-NHEERL-H/ETD/IEG/97/18/01 (Animal 
randomization using a personal computer). The program 
takes all animal weights and ranks them from lowest to 
highest. A group mean and standard deviation is 
calculated for all animals. The number of animals per 
group and the number of groups is entered. The numbers 
of animals available at 1,2, or 3 standard deviations (SD) 
are calculated. The user then selects the lowest SD which 
contains the required number of animals for the study. All 
outliers are eliminated. Additional animals are then 
eliminated to fit into the required number for the study. 
Animals are then randomized by weight into the required 
groups. All animals are accounted for and reasons why 
they were not selected are displayed. The weight 
randomization program was used to identify the groups of 
4 mice which are housed together in a single plastic cage. 
Within each cage, mice were individually identified by 1 
to 4 marks applied to the base of the tail with a Sharpie 
permanent ink marker (Sanford, Bellwood IL). Different 
experimental groups were identified with different colors 
(e.g. saline control mice - green marks, etc.). In addition 
the cage cards were marked to identify the experimental 
group. Marks remained evident for at least two days 
which was long enough to identify mice at 24 hr 
termination points. In cases where mice were killed more 
than 2 days after the initial marking, the tails were 
remarked where necessary because of excessive fading. 
Mice were weighed at the time of randomization, again 
immediately before exposure if randomization occurred 
before the day of exposure, and whenever one group of 
mice was killed. 

G. Toxicological Endpoints: Experimental Design. 

The toxicity of WTC PM, 5 samples was assessed in 
three separate experiments, designated Experiments A, B, 
and C. Experiment A was designed to study the dose- 
response characteristics of the pooled sample of WTC PM 
(WTCX) in comparison with ROFA (toxic control), MSH 
(low toxicity control), and saline vehicle control. 
Experiment B was designed to study the responses 
associated with nose-only inhalation exposure of WTC3 
PM 2 5 in comparison with the responses of mice exposed 
to air only. Experiment C was designed to compare toxic 
responses of WTC PM 2 5 from individual sites with each 
other and with NIST 1649a. 

In all experiments, a group size of 8 was selected 


8 




based on scientific judgement and experience with the 
typical variability of collected data (except the first part of 
experiment A, where n = 12; see Results - Experiment A - 
BAL parameters for explanation). Endpoints were 
analyzed in a total of 388 mice in all 3 experiments. In 
general, the endpoints were chosen to assess pulmonary 
function impairment, lung injury and inflammation, and 
pathological manifestations of respiratory tract injury. 
Experiments utilizing oropharyngeal aspiration (A and C) 
were emphasized over inhalation experiments (B) for 
several reasons: 1 ) the quantities of samples available for 
study were generally limited, and aspiration requires much 
less material ( 10-100 mg) than inhalation (10 g preferred); 
2) aspiration delivers a precise quantity of PM to the lung 
at a specific time point, while the inhaled dose is more 
difficult to predict or quantify; 3) inhalation exposure 
studies are labor intensive and therefore fewer 
comparative analyses of the WTC PM 25 could be 
accomplished in the available time frame compared with 
studies utilizing oropharyngeal aspiration, and 4) 
oropharyngeal aspiration (equivalent to intratracheal 
instillation) is specifically recommended in evaluation of 
panels of test materials for their relative potential to 
produce toxicity (Driscoll et al., 2000). 

/. Experiment A. In 5 sub-experiments, groups of 
female CD-I mice were exposed to pooled WTC PM 25 
sample X (10, 31.6, or 100 pg), MSH (100 pg), ROFA 
(10 or 100 pg), or saline vehicle control by oropharyngeal 
aspiration on day zero. The dose of 31.6 pg represents the 
half-log difference between 10 and 100 pg (i.e. 10 ' ^ = 
31.6). The high dose of 100 pg was selected based on our 
experience that at this dose nearly all PM samples will 
induce at least a mild inflammatory or physiological 
response; any sample that does not induce any response at 
all at this dose can be judged to possess low toxicity. 
Doses higher than 100 pg in the mouse may be of 
questionable relevance due to the potential for artifactual 
local inflammatory responses in response to bolus 
administration (Driscoll et al., 2000). Four mice per 
sample group were tested within each sub-experiment (n 
= 28 per sub-experiment; total experiment A: n = 140). 

In sub-experiments Al, A2, and A5 (total n = 12 mice 
per sample group), airway responses to aspiration of the 
PM samples was assessed by comparison of breathing 
parameters just before and after aspiration (see method 
below). On day 1, diffusing capacity of the lung for 
carbon monoxide (DECO) was assessed, and mice were 
then killed and bronchoalveolar lavage (BAL) fluid cells, 
proteins, and enzymes were recovered and quantified to 
assess lung injury and inflammation. 

In sub-experiments A3 and A4 (n = 8 mice per sample 


group), airway responsiveness to methacholine (Mch) 
aerosol was determined on day 1. Mice were then killed, 
and lungs were removed and fixed for histopathological 
assessment. Airway hyperresponsiveness to nonspecific 
bronchoconstrictive agents such as Mch is a primary 
feature of asthma (Sears, 1997) as well as reactive airways 
dysfunction syndrome (RADS) which develops after high- 
level occupational exposure to irritant gases, fumes, or 
smoke (Gautrin et al., 1999). Induction of this condition 
by PM in nonallergic normal mice can be considered as a 
marker of respiratory tract injury. 

2. Experiment B. Two groups of female CD-I mice 
were exposed in nose-only inhalation exposure tubes one 
time to a PM 2 5 sample (WTC3) or air only for 5 hr (n = 48 
per exposure group; total experiment B: n = 96). This 
WTC3 sample was derived from a sieved but not 
previously fractionated PM 53 sample of WTC3 by 
aerodynamic size-separation during exposure. Although 
some irritant responses are transitory and therefore would 
be best measured during exposure (Costa and Schelegle, 

1999), we do not currently possess the recently developed 
technology (e.g. Buxco Electronics, Sharon, CT or CH 
Technologies, Westwood, NJ) which allows some 
respiratory parameters to be measured during nose-only 
exposures. Therefore, breathing parameters were 
compared just before and after inhalation exposure (n = 12 
per group). On days 1, 3, and 6 after the exposure, 16 
mice from each group were assessed for DLCO and BAL 
parameters (n = 8 ) or responsiveness to Mch aerosol and 
lung and nasal histopathology (n = 8 ). 

3. Experiment C. In 2 sub-experiments, groups of 
female CD-I mice were exposed by oropharyngeal 
aspiration to 100 pg of PM from one of 7 individual WTC 
sample sites, to 100 pg of NIST 1649a (referred to as 
NIST hereafter), or to saline vehicle only. In sub¬ 
experiment Cl, mice were exposed to WTC 8 , WTC 13, 
WTCF, NIST, or saline. In sub-experiment C2, mice were 
exposed to WTCII, WTCB, WTCC, WTCE, or saline. 
On days 1 and 3, mice were assessed for responsiveness to 
Mch aerosol, BAL parameters, and lung histopathology (n 
= 8 per group per time point, except saline sub-experiment 
C2: n = 4 per time point; total sub-experiment C1: n = 80, 
total sub-experiment C2: n = 72, total experiment C: n = 
152). 

H. Oropharyngeal Aspiration of PM Samples. 

A Sartorius model AC211S analytical balance 
(Edgewood, NY; audited by Research Triangle Institute, 
Research Triangle Park, NC) was used to weigh PM 
samples for oropharyngeal aspiration. The operation of 
the balance was tested by weighing calibrated weights 


9 





before and after weighing samples each day (Class U 
calibrated weights, Denver Instrument Company, Arvada, 
CO). PM samples were allowed to come to room 
temperature from 4 °C before weighing. Sterile 2.5 ml 
glass vials or 5 ml polystyrene snap cap vials were used to 
weigh and resuspend PM samples. Vials weights were 
tared, and a sterilized stainless steel spatula was used to 
transfer sample to the weighing vial, and the sample was 
weighed. The sample was then resuspended with sterile 
saline (Sigma S-8776 single use vials, lot 128H2310) 
using a calibrated Rainin Pipetman at a concentration of 2 
mg/ml. All mice aspirated a volume of 50 pi. Samples 
were vortexed and used straight (100 pg dose) or diluted 
as necessary (to 0.632 mg/ml or 0.2 mg/ml for 31.6 pg or 
10 pg doses, respectively). All samples were sonicated for 
2-4 minutes at 22 °C (Branson model 3210R-DTH, 
Danbury, CT) prior to oropharyngeal aspiration. 

Mice randomized into different exposure groups were 
anesthetized in a 2.7 L plexiglass chamber by passing 
house air through an aerator containing methoxyflurane 
(Metofane; Mallinckrodt, Mundelein, IL). The vapor 
induced rapid anesthesia, at which time the mouse was 
taken out of the chamber and placed on an aspiration 
platform. The tongue was gently pulled back and held 
with a forceps, and 50 pi of PM suspension or saline alone 
was pipetted in the back of oropharyngeal region using a 
200 pi tip. The tongue was held until the animal was 
forced to aspirate the sample, and placed back in its cage. 
Mice recovered within 5 or 10 minutes of this procedure. 
This technique is equivalent to intratracheal instillation in 
deposition efficiency (Foster et al., 2001), and several 
publications describe experiments in which it was 
successfully used (e.g. Dreher et al., 1997, Gavett et al., 
1999, Kodavanti et al., 1998). 

1. Nose-Only Inhalation Exposure. 

In order to assess the effects of WTC PM 2 5 on upper 
respiratory tract responses, mice were exposed to WTC3 
or air only in two separate nose-only inhalation exposure 
chambers. The exposures were conducted for 5 hours in 
52-port nose-only flow-by inhalation chambers (Lab 
Products) on November 27,2001. The exposure time was 
based on practical considerations of the tasks involved on 
the exposure day. The WTC3 sample was a tan powder 
received in a plastic jar, and was desiccated at room 
temperature prior to use. Preliminary exposures were 
conducted on several days prior to exposure to set 
exposure parameters, which indicated that an aerosol 
concentration of 10-15 mg/m^ could be achieved. The 
control chamber and the WTC3 chamber had similar flow 
rates (~ 12 L/min) and received air from the same source. 


The aerosol was generated using a unique exposure system 
which conserves sample by using a carpenter’s chalk line 
to pick up particles from a small Tygon tube dust reservoir 
(illustrated in Ledbetter et al., 1998). The dust is carried 
out through an orifice and blown off the string in a 
discharge head with a high velocity air jet. The particles 
are carried through a particle charge neutralizer and 2.5 p 
cut-point cyclone to remove particles larger than PM 2 5, 
and finally enter the inlet of the nose-only chamber. 

Nose-only exposure tubes were constructed from 50 
ml polypropylene centrifuge tubes with the bottom end 
removed. Mice were randomized into exposure groups as 
described above, and 49 in each group were placed in 
exposure tubes (1 extra per group in case any mice died 
during the exposure). Mice were not acclimated to the 
tubes prior to exposure, since stress may be an important 
component of the response to WTC PM. In order to 
measure immediate airway responses to air or WTC3 
sample and also to handle the large number of mice, the 
control air exposure was begun 1 hour before the WTC3 
exposure. 

Dust concentration was determined gravimetrically on 
5 Teflon filters (45 mm diameter with 1 p pore size) taken 
at a sample flow rate of approximately 0.24 L/min. The 
filters were weighed just prior to and after sampling using 
a Cahn C-30 balance housed in a controlled temperature 
and humidity enclosure. Real-time PM concentration was 
achieved with an aerosol monitor (Dust Track, TSl Inc., 
St. Paul, MN) on the chamber exhaust. The particle size 
was determined gravimetrically using a Mercer Cascade 
Impactor (Intox Products, Albuquerque, NM). On 
November 29, 2001, the exposure system was restarted 
and 7 Teflon and 3 polycarbonate filters were taken for 
chemical analysis. On December 17, 2001, 18 
polycarbonate filters (9 WTC3 and 9 blanks) were 
collected for chemical analysis and 3 quartz filters were 
collected for carbon analysis. 

J. Respiratory Responses Assessed by Whole Body 

Plethysmography. 

I. Immediate Airway Responses to PM 2 s Exposure, 
Exposure to PM 25 by oropharyngeal aspiration or by 
inhalation may result in immediate changes in breathing 
parameters. Breathing parameters in unanesthetized 
unrestrained mice were assessed in a 12 -chamber whole 
body plethysmograph system (Buxco Electronics, Sharon, 
CT). The animal chambers have a pneumotach on the roof 
to measure pressure (which is proportional to air flow) 
relative to the pressure in a reference chamber vented to 
the atmosphere. The breath by breath signals are taken by 
the program software to compute respiratory rate 


10 






(frequency, /, breaths / min) and other parameters 
including enhanced pause (PenH). Although PenH is at 
best an indirect measure of flow resistance, it does 
correlate well with lung resistance and reflects changes 
occurring during bronchoconstriction (Hamelmann et al., 
1997), although other responses such as mucus production 
may increase PenH. The convenience of rapidly 
measuring respiratory parameters in twelve mice at once 
was a major consideration in utilizing this technique rather 
than the double plethysmograph or tracheotomized 
ventilated methods which allow direct measures of airways 
resistance and compliance, but are time and labor 
intensive. A protocol was written to record and average 
baseline measurements of mice in calibrated chambers for 
10 min, pause for oropharyngeal aspiration (or stop during 
inhalation exposure), and then resume recording 
measurements for one hour. The time between 
oropharyngeal aspiration and monitoring of responses was 
approximately 6 minutes, while about 20 minutes was 
needed after inhalation exposure to remove mice from 
exposure tubes, weigh them, and transport them to the 
plethysmograph chambers. PenH was automatically 
calculated by the software (and confirmed by examination 
of random data) using expiration time (Te), relaxation time 
(RT), and peak expiratory and inspiratory flows (PEF, 
PIF) according to the following expression: PenH = [(Te- 
RT)/RT] X [PEF/PIFj. Examination of the data after 
exposure showed that utilization of the first 10 or 15 
minutes of the data was not more sensitive in detecting 
changes in respiratory parameters than the entire hour of 
post-exposure monitoring, and therefore responses over 
the whole post-exposure hour were utilized and averaged. 
The percent change in / and PenH after exposure to PM 
was expressed as [(Post-value - Pre-value) / Pre-value] x 
100%. 

2. Airway Responsiveness to Methacholine Aerosol. 
Airway responsiveness to increasing concentrations of 
aerosolized methacholine (Mch) was measured in mice in 
calibrated chambers. After measurement of baseline PenH 
for 5 minutes, saline or Mch in increasing concentrations 
(4, 8, 16, 32, and 64 mg/ml) was nebulized through an 
inlet of the chamber for 1 min. The aerosol drier was 
automatically turned on immediately after the 
aerosolization period for 2 min. Measurements of PenH 
and other parameters were continued for an additional 1, 
2, 3, 4, 8, and 12 minutes after saline or increasing doses 
of Mch, for a total time of 4, 5,6, 7, 11, and 15 minutes (0, 
4, 8, 16, 32, 64 mg/ml Mch, respectively). One minute 
pause periods between aerosolizations allowed time to 
change solutions for nebulization. After subtracting 
baseline values from responses to saline or Mch, the area 


under the curve (PenH AUC; PenH - sec) for these 
recording intervals was calculated using the trapezoid 
method. 

K. Diffusing Capacity of the Lung for Carbon 

Monoxide. 

The ability of the lungs to allow diffusion of gases 
(O 2 , CO 2 ) across the alveolar-capillary barrier is dependent 
on physical properties of the gases and the alveolar¬ 
capillary membrane, and may be limited by perfusion or 
diffusion (Levitzky, 1995). Diffusion limitation may be 
caused by thickening of the alveolar-capillary barrier (e.g. 
by interstitial or alveolar edema). Diffusion of carbon 
monoxide (CO) is limited only by its diffusivity in the 
barrier and by the surface area and thickness of the barrier. 
The diffusing capacity of the lung for CO (DLCO) is 
therefore a useful test of the integrity of the alveolar¬ 
capillary membrane (Levitzky, 1995). 

To determine DLCO rapidly and increase sensitivity 
from individual mice, 4 mice were placed together in a 
single 7.8 L bell jar associated with a Pharmacokinetic 
Uptake System (consisting of an oxygen monitor, flow 
meter, pump, pressure gauge and transducer, mass flow 
controller, and computerized data collection and control 
system). Approximately 6.6 ml of research grade CO 
(99.99%) was injected into the system. The initial 
concentration of CO in the chamber was approximately 
700 ± 10 ppm. CO concentrations were taken every 15 
seconds (Bendix Model 8501-5CA CO Analyzer), and 
continued for approximately 10 minutes. Temperature, 
humidity, airflow, pressure, and oxygen were monitored 
during the test. The DLCO is expressed as the slope of the 
fitted line of [CO] vs. time (ppm/min). 

L. Bronchoalveolar Lavage (BAL). 

Mice were anesthetized with urethane (1.5 g/kg i.p.) 
and killed by exsanguination via severing the renal artery. 
The trachea and lungs were exposed and a 20 g catheter 
was sutured into the trachea. Mice were lavaged with two 
aliquots of Ca^"^, Mg“^, and phenol red-free Hanks’ 
balanced salt solution (HBSS; 35 ml/kg. Life 
Technologies, Bethesda, MD). Approximately 85% of the 
total instilled volume was recovered in all treatment 
groups. The BAL fluid was maintained on ice and 
centrifuged at 360 x g for 10 minutes at 4 °C. 
Supernatants were transferred to a separate tube in order 
to prepare aliquots for biochemical analyses. BAL cells 
were resuspended in 1 ml of HBSS and counted (Coulter 
Zl, Hialeah, FL). Cytospin preparations of BAL cells 
were made for each sample and stained with Wright’s 
Giemsa using an automated slide Stainer (Hematek 2000, 


11 











Elkhart, IN). Cell differentials were performed by one 
person (SHG) counting 500 cells per slide. After lavage, 
the lungs were removed and stored at -80“ C for future 
assays (to be determined). 

Assays for total protein, albumin, lactate 
dehydrogenase (LDH) and N-acetyl-p-D-glucosaminidase 
(NAG) are routine measures of lung injury (Henderson et 
al., 1985) and were carried out on an aliquot of BAL 
supernatant as previously described using a Cobas Fara II 
centrifugal spectrophotometer (Gavett et al., 1997). Four 
other BAL supernatant aliquots were prepared from each 
sample; one of these was supplemented with 10% fetal 
bovine serum to prevent loss of cytokines and other 
proteins in low protein concentration fluids, and the 4 
aliquots from each sample were stored at -80“ C. These 
samples are available for analysis of cytokines and other 
proteins (to be determined). 

M. Histopathology 

7. Lung histopathology. In experiments A and B, 
mice which were tested for Mch responsiveness were 
subsequently assessed for lung histopathology, while in 
experiment C, all mice were tested for Mch responsiveness 
and were lavaged before assessment of lung 
histopathology. Mice were anesthetized with urethane and 
killed as described above for BAL. Lungs were removed 
and fixed by tracheal perfusion in a fume hood with ice 
cold 4% paraformaldehyde at 25 cm pressure for 15 
minutes. The trachea was then tied off and placed in a vial 
of 4% paraformaldehyde at 4 °C. After 24 hours, the lungs 
were drained and placed in phosphate buffered saline at 4 
°C. 

The lungs were transferred to Experimental Pathology 
Laboratories (Research Triangle Park, NC), where fixed 
lungs were processed to paraffin blocks, sectioned at an 
approximate thickness of 5 p, placed on glass slides and 
stained with hematoxylin and eosin (H&E). Longitudinal 
coronal sections were cut on a lateral plane to include 
mainstem bronchi for viewing a maximal amount of lung 
area. Two additional unstained lung sections were 
prepared for future use. Histopathologic observations for 
individual animals in each experiment were tabulated, and 
the degree of severity of inflammatory changes and the 
presence of PM-related pigment were graded on a scale of 
one to five (1 = minimal, 2 = slight/mild, 3 = moderate, 4 
= moderately severe, 5 = severe/high). The pathologist 
knew which animals comprised a group, which group was 
the saline or air-exposed control group, the day after 
treatment, and the doses given to the experimental groups, 
but did not know the identities of the individual PM 
samples other than by a unique number or letter. 


2. Nasal histopathology. Dr. James Wagner of 
Michigan State University (MSU) instructed EPA 
personnel in this procedure, utilized on mice from the 
nose-only inhalation exposure (experiment B). 
Immediately after death, the head of each animal was 
removed from the carcass and both nasal passages were 
fixed by slowly flushing retrograde through the 
nasopharynx with 1-2 ml 4% paraformaldehyde. The 
nasal cavity was then immersed in a large volume of the 
fixative for at least 24h until further processing. The fixed 
nasal cavities were placed in 0.1 M PBS (pH 7.2, 4 °C) 
and shipped overnight to Dr. Wagner at MSU. Nasal 
cavities were decalcified in a 13% solution of formic acid 
for 5 days, and then rinsed in distilled water for Ih. After 
decalcification, three transverse tissue blocks of the nasal 
cavity, cut perpendicular to the hard palate, were selected 
for light microscopic analysis. The first tissue block was 
sectioned from the proximal aspect of the nasal cavity 
immediately posterior to the upper incisor tooth (T1). The 
second transverse tissue block was taken at the level of the 
incisive papilla (T2) and the third and most distal tissue 
block was taken at the level of the second palatial ridge 
(T3). The tissue blocks were embedded in paraffin, and 6 
|im-thick sections were cut from the anterior surface of 
each block. Sections were histochemically stained with 
hematoxylin and eosin for morphologic identification of 
nasal tissues. Nasal tissues (three sections/mouse) from a 
total of 48 mice tested for Mch responsiveness (8 
mice/exposure group/time point) were microscopically 
examined by Dr. Jack Harkema (MSU). Nasal lesions 
were graded on the following scale: 1 = minimal, 2 = 
mild, 3 = moderate, and 4 = marked inflammation. 

N. Statistical Analysis 

All statistical analysis were done using SAS 
procedures (Cary, NC). There were generally three types 
of responses collected: 1) PenH responses recorded 
repeatedly for each animal as area under the curve (AUC) 
for various concentration exposures to Mch; 2) responses 
to DLCO were analyzed from a single response from 4 
animals; and 3) individual measurements measured once 
for each animal as a univariate variable. Experimental 
designs varied with each experiment and each part of an 
experiment. Statistical designs used were replicated 
completely random designs for experiment A. Crossed- 
designs were used for experiment B and C involving 
treatments (TRT) and days (DAY). Randomized block 
designs were used for DLCO experiments. 

When initial multivariate repeated measures analysis 
of variance (MANOVA) test showed significant 
interactions between dose of Mch and TRT or DAY in the 


12 









airway responsiveness studies, univariate linear regression 
was used in all subsequent tests. The models used in 
these regression studies were analysis of covariance 
(COV) with tests for parallelism for each TRT and DAY 
combination. In experiment A, natural logarithms were 
used for both Mch concentration (C) and PenH AUC 
responses. The linear regression Log (PenH AUC) = al + 
b(Log C) reduces to a power function of the form PenH = 
a 2 C‘’. No logarithms were used in Experiments B and C 
due to several negative values resulting after baseline 
adjustments. Techniques similar to ordinary stepwise 
regression were used in COV analyses. Overall test of 
parallelism of regression lines was done first. Subgroups 
of the TRT and DAY combinations were determined to get 
subgroups exhibiting a common slope. Within a subgroup 
with a single slope, subsequent tests were done to 
determine if the means were different (using individual 
contrast tests). Body weight was determined 

repeatedly for animals. Due to animals being removed and 
killed, numbers of mice in each group were different on 
different days after exposure. For days when few animals 
weights were determined, univariate analysis of variance 
(ANOVA) was used to test for TRT effects. For those 
days where most of the weight data occurred, MANOVA 
techniques were used for statistical tests. 

For each TRT and DAY combinations with a 
univariate response, a determination was made if the 
variances could be considered homogeneous. If the 


variance ratios were greater than 10-fold then all of the 
responses were ranked from smallest to largest across all 
TRT and DAY combinations. Then ranks replaced the 
original responses for the univariate ANOVA. Sometimes 
the variances of the ranks for TRT and DAY combinations 
still indicated heterogeneity. Then additional judgment 
was used to help insure that this heterogeneity of variance 
did not affect the overall conclusions. In experiment A, a 
replicate was called DAY. When replication was shown 
to have no significant contribution in the ANOVA results, 
DAY was not included in subsequent ANOVAs. For 
responses with many “zero” values, the residuals from the 
ANOVA were plotted and analyzed by univariate 
techniques to determine if the residuals generally met the 
assumptions required for ANOVA. When interactions 
between TRT and DAY occurred, these were pointed out 
and in some cases further ANOVA were done for each 
DAY. When ranks were used for the response, the ranks 
were regenerated for each day separately. When TRT was 
significant, follow-up comparisons of means were done 
using Tukey’s multiple comparison tests. 

The statistical tests examined only whether groups 
were significantly different from each other. In the 
reporting of the results, for the sake of brevity, groups are 
sometimes referred to as having significantly greater 
values than other groups. These statements should be read 
as groups are significantly different from each other, and 
the mean of one group is greater than the mean of another. 


13 








III. Results 


A. Chemical analysis of solid samples and liquid 

extracts. 

/. Endotoxin and pH levels. The pH of water- 
extracted WTC PM, 5 ranged from 8.88 in WTCE to 10.00 
in WTC 8 (Table 2). The alkaline pH is consistent with 
previous reports of WTC PM (USGS, 2002) and probably 
results from the building materials comprising much of the 
dust (see below). The pH of lyophilized WTC PM 2 5 
reconstituted in unbuffered saline was very close to 
neutral, while MSH was very slightly acidic and ROFA 
was moderately acidic (average 3.74 at 2 mg/ml). It is not 
known why the pH of WTC PMj 5 should be close to 
neutral after reconstitution in saline; perhaps the salt 
neutralizes a basic component of the extract. Endotoxin 
levels in WTC PM 2 5 samples were minimal in comparison 
with other urban PM samples such as NIST 1649a, which 
was also low (Table 2). Several thousand times this level 
of endotoxin caused an acute neutrophilic response in the 
lungs of CD-I mice (Dhingra et al., 2001). The level of 
endotoxin in the samples used in this study would not be 
anticipated to contribute directly to any inflammatory 
response in the lungs. 

2. Elemental and Ion Analysis. The ICP data showed 
that water-soluble calcium and sulfate content amounted 
to 56-63% of the WTC PM 2.5 samples (Table 3). In 
general, the elemental and ion compositions were 
consistent among the different samples tested. ICP data 
for the IM HCl-soluble extracts of WTC PM 2 5 showed an 
additional 1-2 weight percent calcium content. This 
increase may be attributed to calcite or other water- 
insoluble calcium salts which are soluble in IM HCl (see 
below for data on compound analysis). There was no 
evidence of stainless steel contamination from the forceps 
used to handle the WTC PM filters or from the stainless 
steel balls used to size-fractionate the ROFA sample. 

The ICP results for the aerosolized PM 2 5 cut fraction 
of WTC3 generally agree well with those determined by 
XRF (Table 3). Calcium content of acid-extracted WTC3 
was somewhat lower by ICP (20-22%) than calcium 
content of solid WTC3 by XRF (26.6%). This may reflect 
an incomplete extraction in the one hour timeframe of 


sample preparation method for ICP, or the presence of 
other insoluble forms of calcium in the WTC3. The XRF 
values are higher for most other elements, which reflects 
the incomplete dissolution of the WTC3 matrix by the 
weak (water) and moderate (IM HCl) extraction liquids. 
Elements such as magnesium and zinc, which exist in 
compounds more amenable to acid dissolution, agree more 
closely (Weast, 1985; Budavari, 1996). Elements such as 
aluminum, iron, and titanium, which are in the form of 


Table 2. Endotoxin and pH Levels of PM Samples after Water 
Extraction and Resuspension in Saline. 


Sample 

Code'* 

pH in 
water 

Endotoxin 

EU/ml Inhibition 

pH in 
Saline ® 

Water 

5.28 




WTC 8-100 

10.00 

0.50 

none 


WTC 11-100 

9.16 

0.25 

none 


WTC 13-100 

9.47 

0.50 

none 


WTC B-IOO 

9.54 

0.25 

none 


WTC C-100 

9.32 

0.50 

none 


WTCE-100 

8.88 

0.25 

none 


WTC F-lOO 

9.55 

0.50 

none 


NIST-100' 

4.20 

25 

none 


Saline 




6.67 

WTCX-10 




7.38 

WTCX-31.6 




7.38 

WTCX-100 

9.35 



7.36 

MSH-100 




6.61 

ROFA-100 




3.74 


WTCX indicates pooled sample of WTC8, WTCll,WTC13, 
WTCB, WTCC, WTCE, and WTCF. "-100" indicates 100 pg/50 pi 
dose = 2 mg/ml. "-31.6" indicates 31.6 pg/50 pi dose = 0.632 
mg/ml. "-10" indicates 10 pg/50 pi dose = 0.2 mg/ml. 
Water-extracted PM samples were lyophilized and resuspended in 
sterile saline. 

Water used to extract filters. 

‘ NIST Standard Reference Material 1649a (Washington DC TSP 
PM). 

^ Endotoxin levels measured as endotoxin units (EU) per ml water 
extract. Samples were tested for inhibition of the endotoxin assay 
(none was detected). 

' Average of 3-4 measurements. 


14 







Table 3. Elemental and Ion Analysis of WTC2001 Samples 


Sample: 

Diluent: 

No. Analyses: 


WTC 3 


WTCX fDooledl 

WTCB 

MSH 

ROFA 

NIST 

DI HjO 

4 

IM HCl 

4 

none 

5 

DI H,0 

2 

IMHCl 

1 

DI H20 

1 

Dl H20 

2 

IM HCl 

1 

DI H20 

2 

IM HCl 

1 

Dl H20 

1 

Analyte 












S 04 -' 

376488.6 

344439.8 

375300 

439120.3 

379429.4 

432570.7 

955.2 

973.4 

274962.0 

242277.8 

86725.3 

Ca 

184904.4 

218019.2 

265600 

187493.7 

196745.9 

183794.1 

351.5 

1970.8 

18590.8 

19663.0 

12649.5 

Si 



30000 









A1 

1346.3 

4072.2 

9930 

555.7 (*) 

1476.8 

537.1 (*) 

48.4 (*) 

1260.4 

6739.8 

8604.3 

1049.1 

Mg 

1112.9 

5414.1 

6550 

651.5 

2257.6 

354.1 

37.3 

1250.2 

24895.8 

24655.2 

1140.7 

Fe 

150.0 (*) 

2450.4 

6290 

6.7 0 

1098.5 

6.0 (*) 

-0.1 (*) 

1833.4 

763.0 

19512.4 

407.5 

Cl- 

2851.0 


3330 

2699.0 



1103.0 


302.0 


1164.0 

K 



2690 









Zn 

22.7 

1413.1 

1760 

13.4 

410.1 

4.1 

-0.0 (*) 

3.7 

6555.9 

5932.3 

503.9 

Ti 

12.1 

180.9 

1450 

5.2 

41.6 

4.0 

1.3 

17.6 

2.6 

137.5 

1.8 

Na^ 

1290.0 


725 

1139.0 



721.0 


44179.0 


2153.0 

NOj 

938.0 



6496.0 



0.0 


0.0 


7390.0 

P04-' 

0.0 


779 

0.0 



0.0 


0.0 


3317,0 

F 

406.0 



799.0 



0.0 


352.0 


466.0 

Pb 

2.2 

141.3 


0.7 

33.4 

0.4 

0.0 (*) 

0.5 

17.9 

789.6 

1378.7 

Mn 

4.5 

107.9 


1.8 

24.0 

0.7 

3.8 

38.4 

365.0 

458.6 

77.2 

Cu 

5.5 

76.0 


6.4 

21.8 

4.7 

0.6 

6.8 

572.8 

628.4 

83.6 

Ba 

25.6 

75.6 


8.7 

31.0 

7.2 

0.1 (♦) 

5.0 

3.0 

57.5 

11.2 

Sb 

17.7 

43.8 


9.4 

17.4 

7.0 

0.0 (*) 

-0.0 (*) 

1.5 

146.5 

3.5 

NO,- 

38.0 



0.0 



0.0 


0.0 


0.0 

Mo 

1.0 

33.7 


1.8 

3.2 

4.9 

0.1 

0.1 

0.0 (*) 

339.3 

1.8 

Ni 

2.6 

19.7 


4.1 

3.1 

1.8 

0.0 

0.9 

17027.7 

16988.2 

32.2 

Sn 

0.8 

12.1 


1.3 

3.1 

0.1 (*) 

0.0 (*) 

0.0 (*) 

0.0 (*) 

51.0 

0.1 (*) 

Cr 

10.0 



1.4 0 


1.2 0 

0.1 (*) 


6.1 


6.1 

Cd 

0.3 

7.2 


0.7 

1.9 

0.3 

0.0 

0.0 

8.9 

11.9 

19.9 

Be 

-0.0 (*) 

2.7 (*) 


0.3 (♦) 

0.2 (*) 

-0.1 O 

0.0 (*) 

0.0 (*) 

1.4 (*) 

1.7 C) 

0.1 (*) 

Co 

0.2 (*) 

1.6 


0.8 

0.9 

0.1 (*) 

0.0 (*) 

0.4 (*) 

495.0 

510.4 

2.1 

As 

1.3 

-10.4 (*) 


1.1 

-0.4 (*) 

0.3 

0.1 

0.2 

1.7 

106.4 

13.5 

Tl 

0.1 (*) 

-25.5 (*) 


0.5 

-1.2 0 

0.1 (*) 

0.0 (*) 

-0.1 (*) 

0.0 (*) 

-0.2 (*) 

0.3 

V 









1748.6 

35693.6 


NH4^ 

0.0 



0.0 



0.0 


0.0 


25355.0 

Total pg/g: 

569632 

576475 

704404 

639019 

581598 

617299 

3223 

7362 

397593 

376566 

143953 

% Total Mass: 

57.0 

57.6 

70.4 

63.9 

58.2 

61.7 

0.3 

0.7 

39.8 

37.7 

14.4 


“ Results shown are average values for number of analyses indicated, expressed as pg soluble analyte / g solid sample extracted, for deionized (Dl) 
water and IM HCl extractions. Analyte concentrations were detemiined by ICP-MS, except underlined values which were determined by 
ICP-AES, bold values which were determined by ion chromatography, and column labelled none, where solid sample was analyzed by XRF 
(indicated by heavy solid-line box). (*) Value below detection limit. Analytes are arranged in order of decreasing content in WTC 3 sample, 
by whatever analysis provided highest content. 


complex oxides usually in combination with silicon, are 
much less soluble under the acid extraction conditions 
used in this study, and do not agree as well (Weast, 1985; 
Budavari, 1996). The weight-percent ratios of silicon, 
aluminum, magnesium, and iron are in the proportion of 
those found in portland cement, a major component of 


concrete (NIST, 2002; McKetta, 1978). 

Comparison of the water-soluble transition and heavy 
metal content of the WTC PM samples with the control 
dusts shows the overall metal level trend as Mt. St. Helens 
< WTC2001 < NIST < ROFA. ROFA has high levels of 
water-soluble transition metals including vanadium. 


15 














































































Table 4. Carbon fraction analysis of PM Samples in WTC2001 
Study “ 


Sample: 

WTC3 

WTCX 

(pooled) 

WTC B 

MSH 

ROFA 

NIST 

SRM 

1649a 

% Carbon 
Fraction: 







Organic 

6.88 

0.93 

2.11 

0.06 

1.31 

10.82 

Elemental 

0.31 

0.00 

0.01 

0.07 

13.63 

15.10 

Carbonate 

1.39 

0.60 

0.35 

0.00 

1.32 

0.00 

Total 

8.58 

1.53 

2.47 

0.13 

16.26 

25.92 


“ Organic, elemental, and carbonate carbon fractions were analyzed as 
described in text. Results are expressed as percent of total mass of 
sample. 


nickel, and iron which are important in its toxicity 
(Kodavanti et al., 1998). The IM HCl-soluble metal 
content trend is Mt. St. Helens < WTC2001 < ROFA (not 
enough NIST sample was available to run the test). 

3. Carbon analysis. The WTC2001 samples had low 
total carbon content, in the range of 1.5-8.5% (Table 4), in 
comparison with control samples such as NIST (26%) and 
ROFA (16%). MSH had almost no carbon, as expected 
from this crustal PM sample. The WTC3 sample used in 
the inhalation study had about 4 times as much carbon as 


Table 5. XRD Analysis of Compounds Present in WTC 3 
Sample ® 


ICDD 

Number 

Formula 

Mineral 

Name 

Relative 

Amount 

05-0586 

CaCOj 

Calcite 

Major 

33-0311 

CaS 04 - 2 H 20 

Gypsum 

Major 

41-0224 

CaSO40.5H2O 

Bassanite 

Minor 

46-1045 

SiOj 

Quartz 

Minor 


“ Analysis showed about half crystalline materials (50.6% above 
background), and the remainder was amorphous. After smoothing 
and subtracting background, evaluation software (MDI Jade 5) was 
used to match patterns with library available from International 
Centre for Diffraction Data Powder Diffraction File, release 2000. 

the other two WTC samples. This result may be due to 
differences in the method by which the samples were 
isolated (physical separation vs. aqueous extraction and 
lyophilization) or may simply be due to variability in 
carbon content of samples from different locations. 
Despite the variation in total carbon content of WTC PM 
samples, the ratios of elemental, organic, and carbonate 
carbon were similar. Elements not listed in Table 3 or 4 
(-30% of total mass) are likely O and H from adsorbed 

water and O, H, and N from 
organic or inorganic compounds. 

4. Compound analysis by 
XRD. XRD analysis of WTC3 
PM 53 (before size segregation by 
the inhalation exposure system) 
showed a complex pattern 
containing 25 peaks, indicating the 
presence of several crystalline 
materials. The peak area above the 
background curve was 50.6%. The 
49.4% below the curve indicated 
that WTC3 consisted of about half 
amorphous materials. Four 
patterns were identified as being 
consistent with peaks identified in 
the dust. Figure 3 shows the XRD 
spectra of WTC3 and those of the 
matched compounds. Two 
compounds were identified as 
major constituents (calcium 
carbonate (calcite) and calcium 
sulfate dihydrate (gypsum)), and 
two were identified as minor 
constituents (bassanite and quartz. 
Table 5). The XRD data are 


' • ■ • I. I....... I....... I... I... I... I..., 


1000 - 


^ 750 

ZJ 

o 

5 500 

c 
a> 


250 


1020118A.MDI1 WTC dust, as raceived 


rU.S. EPA 









, 


33*0311> Gypsum • CaS04!2H20 

..— 1 - ■ , .... . ^ 


i 

. i ^ .1 

41-0224> Bassanite - CaS04!0.5H20 

» ■ ‘ . . . . W . . . . t ■ ■ ■ 1 J 1 . . XA ^ . 4 . » . ^ 1 1 t t ..... 


05*1)586 > Calcite - CaC03 

1 - : .... . . 

■ r" T" r ' r T" 1 ' 1 1 1 

46-1045 > Oijaitz - Si 02 

■T'‘T“T"‘f r‘'T’T'"’T“T‘’r ■ V ■ y ■ r ' ‘ r “* I 1 ■f T'"l ■! 1 T T 


2-Thetan 

lXRD|Administratorl<cADATASCAmDATA01> Wednesday. Feb 06. 2002 04:13p (MOI/JADH 


Figure 3. X-ray diffraction (XRD) analysis of WTC 3 sample (PM 53 ) used in nose-only 
inhalation exposure study (Experiment B). Peaks were collected in the range, 20 = 5 - 85°. 
Collection software used was Materials Data, Inc. Datascan, version 3.2 


16 









































Figure 4. SEM/EDX results from water-extracted lyophilized WTC PM samples. The upper-left 
quadrant of each photomicrograph shows a field of view with the particle of interest within the 
smaller square in that field. The upper-right quadrant shows a zoomed-in view of the feature (the 
area from within the square in the upper-left quadrant), and the lower half shows the elemental 
spectrum acquired with the electron beam centered on the small (barely visible) square in the 
zoomed-in view. A. Example of Ca-S crystal which dominated the samples. B. Example of fine 
particle aggregate which was prominent in the samples. C. Example of fiber found in the samples. 
D. Example of metallic particle within fine particle aggregate. 


consistent with the ICP data which show water-soluble 
calcium and sulfate in the same proportions as gypsum. 
Gypsum is completely water-soluble at the solid/liquid 
ratio of the extraction conditions used in the ICP analysis, 
while calcite is not water-soluble. The sample of MSH 
was also analyzed by XRD and the results were consistent 
with those previously reported (Graham et al., 1985; data 
not shown). 

5. SEM/EDX analysis. Water-extracted and 
lyophilized WTC PM samples were dominated by 
snowflake-like crystals composed of calcium and sulfur 
(Figure 4A). Aggregates of fine particles composed of 
various combinations of Mg, Al, Si, S, and Ca were also 


prominent (Figure 4B). Fibers approximately 1 pm in 
diameter were found in most of the samples and had a 
composition similar to the fine particle aggregates (Figure 
4C). Metallic particles (mostly Ti and Fe, though Zn, Pb, 
Ba, and Cu were also found) were found typically as 
inclusions in the large fine particle aggregates (Figure 4D). 
The crystals and aggregates were likely not original to the 
bulk sample but were formed as a result of the aqueous 
extraction process. 

SEM/EDX analysis of the aerosolized PM 2 5 cut 
fraction of WTC3 showed the same overall chemistry as 
the extracted and lyophilized WTC PM samples: the 
majority of particles were composed of Ca or Ca-S, some 


17 
















Figure 5. Particle types found in the WTC3 sample used in the nose-only inhalation exposure 
(Experiment B). A. Example of Ca-S particle which was prominent in the sample. B. Example of 
Ca particle which was prominent in the sample. 


also containing Si. Some representative particles are 
shown in Figures 5 A and 5B. In contrast to the crystals 
and aggregates of the bulk solid samples as described 
above and shown in Figure 4, the particles of WTC3 are 
small, typically about 1 pm, with rough, irregular features. 
The different form of the Ca-based particles in WTC3 
reflects the dry size segregation of the inhalation exposure 
system. Particles with other compositions were found 
with far less frequency. These included particles 
composed of Fe, C, and Sb-Zn (one example found). One 


or more possible asbestos fibers (Mg-Si composition) were 
also found, however polarized light microscopy rather than 
SEM/EDX is the preferred method for identifying 
asbestos. SEM/EDX analysis was also performed on 
MSH, NIST 1649a, and ROFA and showed results 
typically found in previous analyses (data not shown). 

6. Summary. WTC PM samples consist primarily of 
construction materials from the fallen-down WTC 
buildings. The bulk of the WTC PM samples are calcium- 
based compounds, specifically calcium sulfate (gypsum) 


Table 6. Experiment A: Body Weights and Immediate Airway Responses. 


Group 

B.Wt. d 0 

R 

B.Wt. d 1 

S 

Breathing Frequency (min"') 

PenH tunitless) 

Pre- 

Post- 

% increase 

Pre- 

Post- 

% increase 

Saline 

25.8 

24.9 

492.3 

348.1 

- 29.7 

0.73 

0.96 

23.9 


0.4 

0.4 

11.5 

25.6 

4.2 

0.08 

0.17 

8.3 

MSH-100 

25.5 

25.0 

474.0 

320.6 

- 31.7 

0.92 

1.25 

40.5 


0.4 

0.3 

13.5 

23.0 

5.2 

0.13 

0.18 

14.8 

ROFA-10 

25.6 

24.3 

492.0 

343.5 

- 29.7 

0.74 

1.05 

42.3 


0.4 

0.6 

14.3 

18.6 

4.1 

0.11 

0.17 

13.5 

ROFA-100 

25.2 

25.2 

461.0 

307.0 

- 33.4 

0.88 

1.51 

76.4 


0.5 

0.4 

11.9 

18.7 

3.7 

0.10 

0.20 

18.6 

WTCX-10 

25.2 

24.3 

467.2 

322.4 

- 31.1 

0.85 

1.14 

52.4 


0.6 

0.5 

14.7 

22.5 

4.1 

0.15 

0.18 

21.8 

WTCX-31.6 

25.3 

24.7 

476.8 

348.5 

- 26.3 

0.86 

1.14 

26.8 


0.5 

0.5 

15.5 

18.9 

4.3 

0.18 

0.34 

15.6 

WTCX-100 

25.6 

25.1 

486.8 

325.3 

- 33.1 

0.79 

1.11 

40.7 


0.6 

0.4 

14.1 

26.6 

5.1 

0.08 

0.16 

11.8 


Values shown are means (in bold) and SEM immediately below means (n=12 per group). Body weight (B. Wt.) 
was measured in the morning. Respiratory parameters were measured immediately before (Pre-) and after (Post-) 
oropharyngeal aspiration of dust samples or saline on day 0. Values within solid-line boxes indicate significantly 
greater values in ROFA-100 mice vs. Saline mice (f* < 0.05). 


18 



















and calcium carbonate (calcite). Together these salts 
compose about two-thirds of WTC PM 53 on a weight 
percent basis. Given the prevalent use of gypsum in 
ceiling tiles and wallboard, and the ease with which these 
building materials can be crumbled into dust, the high 
gypsum content is reasonable. Elemental analysis 
indicates that the other main components of WTC PM are 
construction materials such as cement and concrete 
aggregate. The elemental composition of WTC PMj 5 was 
consistent with that of sieved unffactionated WTC PM 53 
(as WTC3, Table 5). Carbon and metal content of the 
WTC samples were low, as expected from crustal-derived 
building materials (McKetta, 1978). A more complete 
chemical and physical analysis of dust samples has 
recently been reported by the U.S. Geological Survey 
(USGS, 2002). In that study, dust samples were collected 
from undisturbed locations within a 1 km radius of the 
WTC site on September 17 and 18,2001 (after the rain of 
September 14,2001). The present report generally agrees 
with the findings of the USGS study, including the 
alkaline nature of the WTC PM extracts. 

B. Experiment A: Dose-Response Relationships of 
WTC PMj 5 

I. Body weights and immediate airway responses. 


Table 7. Experiment A: Diffusing Capacity of the Lung for 
Carbon Monoxide (DLCO) “ 


Sub-experiment: 

Date: 

Treatment Group 

A1 

11/6/01 

A2 

11/8/01 

A5 

12/6/01 

Mean 

n = 3 

SEM 

Saline 

-3.474 

-3.610 

-4.510 

-3.865 

0.325 

MSH-100 

-3.734 

-3.944 

-4.338 

-4.005 

0.177 

ROFA-10 

-3.904 

-3.597 

-3.928 

-3.810 

0.107 

ROFA-100 

-4.015 

-3.089 

-4.293 

-3.799 

0.364 

WTCX-10 

-2.759 

-3.811 

-4.301 

-3.624 

0.455 

WTCX-31.6 

-4.051 

-3.338 

-4.014 

-3.801 

0.232 

WTCX-100 

-3.551 

-4.433 

-4.299 

-4.094 

0.275 


* Diffusing capacity of the lung for carbon monoxide was determined 
one day after exposure on four mice from each treatment group, placed 
together in a single bell jar, in order to rapidly assess DLCO and 
reduce individual variability. Values shown are slopes of chamber 
[CO] vs. time (ppm/min), after subtraction of value from empty 
chamber. No significant differences among any treatment groups were 
detected. 

Mice were exposed by oropharyngeal aspiration with 
PM 2 5 samples of pooled WTC sample X (10, 31.6, or 100 
jig), MSH (100 |ig), ROFA (10 or 100 pg), or saline on 
day zero. In sub-experiments A1, A2, and A5, immediate 
airway responses were determined on day 0, and DLCO 


Tables. Experiment A: BAL Parameters (Day 1 ). ® 


Group 

BAL Cell Number (x 10 ") 

Protein 

LDH 

Albumin 

NAG 


Mac 

Eos 

Neut 

Lym 

Ug/ml 

U/L 

Ufi/ml 

U/L 

Saline 

18.80 

0.02 

0.23 

0.10 

155.2 

29.8 

21.8 

2.2 


4.05 

0.02 

0.16 

0.02 

6.8 

2.1 

1.2 

0.4 

MSH-100 

27.78 

0.07 

0.78 1 

0.18 

168.8 

27.8 

22.3 

2.0 


5.96 

0.03 

__Q.^ J 

0.05 

8.8 

1.7 

1.2 

0.4 

ROFA-10 

27.36 

0.00 

0.21 

0.18 

157.9 

32.3 

20.8 

3.1 


2.70 

0.00 

0.05 

0.07 

5.3 

1.2 

0.9 

0.4 

ROFA-100 

28.69 

0.16 

13.18 

0.42 

279.5 

93.2 

39.2 

7.9 


3.98 

0.06 

2.44 

0.09 

16.8 

10.3 

2.8 

1.2 

WTCX-10 

22.28 

0.00 

0.09 

0.14 

153.7 

30.4 

20.8 

1.9 


4.03 

0.00 

0.02 

0.04 

4.3 

1.8 

0.8 

0.3 

WTCX-31.6 

31.36 

0.01 

0.37 

0.21 

160.2 

33.6 

21.7 

1.8 


7.73 

0.01 

0.14 

0.03 

6.3 

1.6 

1.3 

0.2 

WTCX-100 

20.48 

0.06 j 

1.43 1 

0.23 

161.4 

33.7 

21.3 

2.3 


1.73 

0.03 

._0-24 J 

0.04 

4.8 

2.1 

1.0 

0.3 


“ Values shown are means (in bold) and SEM immediately below means (n=l 2 per group). Bronchoalveolar lavage 
(BAL) cell numbers and proteins were recovered 1 day after exposure. Cell types shown are macrophages and 
monocytes (Mac), eosinophils (Eos), neutrophils (Neut), and lymphocytes (Lym). Total protein, lactate 
dehydrogenase (LDH), albumin, and N-acetyl-P-D-glucosaminidase (NAG) were measured in BAL fluid 
supernatant. Values within solid-line boxes indicate significantly greater values in ROFA-100 mice vs. Saline 
mice {P < 0.05). Values with dashed-line boxes indicate significantly greater values (P < 0.05) compared with 
Saline mice (excluding ROFA-100 data which generally had much larger variances than other groups). 


19 























Mac 


Neut 





I I Saline 
CZZI^MSH- 100 
ESSSESEES ROFA - 10 
■■■ROFA-100 
izzzzza WTCX-10 
ESS3 WTCX-31.6 
E2S23 WTCX -100 


b 



1.0-1 

0 . 8 - 


o 

^ 0 . 6 - 
CA 


C 0-4H 


0.2H 

0.0 


Eos 


Lym 


L - 1 Saline 

dHH MSH- 100 
mm ROFA - 10 
■■■ROFA - 100 
fZZZZZ2 WTCX - 10 
ESS3 WTCX-31.6 
Ema WTCX -100 



1 . 0-1 


0 . 8 - 


o 

^ 0 . 6 - 
g 0.4H 


0.2H 

0.0 


L . i Saline 

C=1MSH-100 
ROFA - 10 
1^™ ROFA-100 
IZZZZZ3 WTCX - 10 
ESSS3 WTCX-31.6 


ESSSa WTCX - 100 



Figure 6. Experiment A. Bronchoalveolar lavage cell numbers recovered from mice one day after exposure by intratracheal 
instillation to PM samples in saline or saline vehicle alone. Values shown are means and SEM (n=12 per group). Cell types shown 
are macrophages and monocytes (Mac), eosinophils (Eos), neutrophils (Neut), and lymphocytes (Lym). ^ P< 0.05 vs. Saline group. 
*‘P< 0.05 vs. Saline group (comparison of rank values) after exclusion of ROFA-100 data which had much larger variances than other 
groups. 


and BAL parameters were determined on day 1. There 
were no significant differences in body weights of the 
seven groups on day 0 or day 1 (Table 6 ). Ventilatory 
parameters were assessed in mice immediately before and 
after exposure. There were no differences among groups 
in breathing frequency, but mice exposed to the 100 pg 
dose of ROFA (ROFA-100) had a significant increase in 
PenH immediately after exposure in comparison with 
saline control mice (Table 6 ). There were no significant 
changes in immediate responses in mice exposed to any 
dose of WTCX. 

2. DLCO. The diffusing capacity of the lung for 
carbon monoxide was determined 24 hr after 
oropharyngeal aspiration on groups of 4 mice from the 
same exposure group together in the testing chamber. 
There were no significant differences in DLCO among any 


of the groups of mice, which would be indicated by a 
slower uptake of CO and a reduced slope (Table 7). These 
data indicate that none of the PM samples caused injury 
severe enough to significantly reduce gas exchange at the 
alveolar-capillary barrier. 

3. BAL parameters. Bronchoalveolar lavage 
parameters were determined immediately after testing for 
DLCO. Originally, we planned to do just two sub¬ 
experiments for this part of Experiment A. However, we 
noted that there was significant variation in the total cell 
numbers recovered from the 4 saline control mice in each 
of sub-experiments A1 and A2 (average of 7 x lO"^ vs. 33 
X 10'’, respectively). There was no evidence of any 
infection (in both cases 99% of BAL cells recovered from 
control mice were alveolar macrophages (AMs)), mice 
came from the same shipment in the same week, and no 


20 




























































































































Protein 



1 I Saline 
C=] MSH- 100 
ROFA - 10 
■■■ ROFA - 100 
IZZZZZI WTCX - 10 
ESSS3 WTCX-31.6 
Esma WTCX -100 



Albumin l=ZZI Saline 



MSH - 100 
mma ROFA -10 
■■■ ROFA -100 
VTTTn WTCX - 10 
WTCX-31.6 
Esma WTCX -100 




LDH 



120-1 


80- 




I I Saline 

MSH - 100 
mmsi ROFA -10 
ROFA -100 
IZZZZZ3 WTCX - 10 
ESSS3 WTCX-31.6 
Emsa WTCX -100 



NAG 

I I Saline 

a CZIZZ3 MSH-100 


ROFA -10 



Figure 7. Experiment A. Values for total protein, lactate dehydrogenase (LDH), albumin, and N-acetyl-b-D-glucosaminidase (NAG) 
were measured in bronchoalveolar lavage fluid supernatants recovered from mice one day after exposure by intratracheal instillation 
to PM samples in saline or saline vehicle alone. Values shown are means and SEM (n=12 per group). ^ P< 0.001 vs. Saline group. 


other reason could be deduced for the difference. 
Consequently, we performed a third sub-experiment to 
examine these endpoints and increase the number of mice 
per group (sub-experiment A5). The average total cell 
number recovered from saline control mice in A5 was 18 
X 10"^ (97% AMs) - about in the middle between A1 and 
A2. All data shown is combined from the 3 sub¬ 
experiments. Due to the high variance of data in the 
ROFA-100 group, we judged that it was necessary to 
compare ROFA-100 data alone vs. saline control data. 
Other comparisons were made between the saline control 
group and the other groups after excluding ROFA-100 
data. Significant increases in neutrophils, eosinophils, and 
lymphocytes were found in ROFA-100 mice compared 
with saline control mice (Table 8). Neutrophils comprised 
31% of total BAL cells in ROFA-100 mice (Figure 6). 
After excluding the ROFA-100 data, significant 
differences in neutrophil numbers were found between the 


saline control group and both the MSH-100 group and the 
WTCX-100 group (Table 8, Figure 6; P < 0.05). 
Neutrophils comprised about 7% of total BAL cells in the 
WTCX-100 group, but only about 1% or less in the 
WTCX-31.6 and WTCX-10 groups. 

Levels of proteins and enzymes were measured in the 
BAL supernatant to assess lung damage. Both total protein 
and albumin are increased after damage to the alveolar 
epithelial barrier (Henderson et al., 1985). Lactate 
dehydrogenase (LDH) is a cytoplasmic enzyme which is 
released by dead or dying cells, while N-acetyl-P-D- 
glucosaminidase (NAG) is indicative of lysosomal enzyme 
release (Henderson et al., 1985). All of these parameters 
were significantly increased in the ROFA-100 group in 
comparison to saline control mice (Table 8). Total protein 
and albumin were both increased about 80% compared to 
saline, while LDH was increased 3-fold and NAG almost 
4-fold (Figure 7). No significant changes in BAL proteins 


21 


















































































































































































Table 9. Experiment A: Body Weights, Baseline PenH, and Responsiveness to Methacholine Aerosol 


Group 

B.Wt. dO 

(g) 

B.Wt. d 1 

(g) 

Baseline 

PenH 


Dose Mch (me/ml) and PenH AUC (PenH - sec) 


0 

4 

8 

16 

32 

64 

Saline 

24.78 

23.30 

0.97 

0.5 

39.1 

54.0 

142.3 

309.0 

1249.9 


0.42 

0.65 

0.13 

0.5 

8.4 

8.9 

28.5 

30.6 

360.7 

MSH-100 

24.82 

23.80 

1.10 

2.4 

36.4 

99.2 

177.9 

437.9 

1173.3 


0.51 

0.45 

0.12 

0.3 

5.5 

22.5 

23.7 

54.3 

358.3 

ROF A-10 

24.99 

24.55 

0.81 

15.5 

42.4 

114.6 

205.5 

432.4 

1182.5 


0.43 

0.45 

0.10 

3.2 

8.9 

11.3 

56.1 

88.5 

294.2 

ROFA-100 

24.86 

24.60 

0.91 

6.1 

36.7 

118.2 

242.1 

642.2 

2190.9 


0.44 

0.45 

0.11 

6.3 

4.1 

34.1 

37.2 

94.0 

706.4 

WTCX-10 

24.96 

24.44 

0.92 

18.5 

43.9 

88.3 

169.1 

281.1 

968.4 


0.38 

0.28 

0.11 

6.4 

11.6 

16.2 

52.3 

35.5 

129.9 

WTCX-31.6 

24.56 

23.73 

0.85 

15.3 

38.3 

55.2 

114.4 

249.2 

923.3 


0.46 

0.51 

0.11 

3.4 

4.8 

8.8 

7.0 

39.8 

174.2 

WTCX-100 

24.68 

24.14 

1.04 

9.5 

67.4 

208.3 

397.3 

2265.0 

4009.1 


0.44 

0.42 

0.12 

8.2 

11.3 

47.6 

61.4 

260.7 

580.1 


“ Values shown are means (in bold) and SEM immediately below means (n=8 per group). Body weight (B. Wt.) was measured 
in the morning (no significant differences were found). No significant differences were found in baseline PenH (enhanced 
pause; unitless) on day 1. Methacholine aerosol (Mch) was then administered (see Methods for details) at the indicated doses, 
and the airway response was calculated as the area under the curve (AUC) of the PenH response over time in seconds. See 
Figure 8 for description of statistical analysis of PenH AUC data. 


and enzymes were found in any of the other PM exposure 
groups relative to saline controls. In general, the 
inflammatory response in the WTCX-100 group can be 
considered to be quite mild considering the fairly high 
dose. 

4. Responsiveness to methacholine aerosol. In sub¬ 
experiments A3 and A4, the same groups of mice were 
exposed and the same time points were examined as 
described for sub-experiments AI, A2, and A5, but 
different endpoints were examined. There were no 
differences in body weights on day 0 or day 1 among the 
7 groups of mice (Table 9). On day I, there was no 
difference in baseline PenH values (immediately before 
Mch aerosol) among the 7 groups. Responsiveness to 
increasing concentrations of Mch aerosol was assessed and 
quantified by integrating the area under the PenH - time 
curve (PenH AUC; Table 9). In order to assess overall 
responsiveness and account for variability, power function 
equations were fit to the PenH AUC vs. [Mch] data for 
each group (Figure 8). The analysis showed that the 
Saline, MSH, ROFA-10, WTCX-10, and WTCX-31.6 
groups could all be modeled with a common power 
function exponent (1.157). It is important to note that 
once the lines were determined to come from groups with 
a common exponent, the lines for these 5 groups were fit 
simultaneously, resulting in fitted equations that did not fit 


as well as an individual line would fit the group-specific 
data. The responses to saline or individual doses of Mch 
aerosol are not as important as the fitted line describing 
the groups. Among these 5 groups, ROFA-10 mice had a 
small but significant increase in the coefficient of the 
equation vs. the Saline group (P = 0.03). The ROFA-100 
and WTCX-100 groups could be modeled with a power 
function with a significantly different exponent (1.471; P 
= 0.001) vs. the common exponent of the other 5 groups, 
indicating that these 2 groups are hyperresponsive 
compared with the other 5 groups. In addition, the 
coefficient for the WTCX-100 group was significantly 
different Ifom and greater than that of the ROFA-100 
group (P = 0.0001), showing that mice exposed to the 100 
pg dose of WTCX were more reactive to Mch than the 
ROFA-100 group. 

5. Lung histopathology. Following tests for airway 
responsiveness to Mch aerosol, mice from sub¬ 
experiments A3 and A4 were killed and assessed for 
pathological changes in the lungs. No remarkable findings 
were observed in the lungs of the saline control group 
(Table 10). In both the MSH-100 and ROFA-100 groups, 
focal subacute bronchiolar inflammation was found at 
similar incidences and average severity, which was 
minimal (average score: MSH-100 = 0.8; ROFA-100 = 
1.0). The ROF A-10 group had a lower average severity 


22 















Saline 


MSH 



Figure 8. Experiment A: Airway responsiveness to methacholine aerosol challenge in mice exposed to PM samples or saline vehicle 
and tested one day later (n = 8/group; data shown are mean + SEM). Power function equations were fit to the data. Saline, MSH, 
ROFAIO, WTCXIO, and WTCX31.6 equation exponents were not significantly different. Significantly different exponent vs. 
common Saline, MSH, ROFAIO, WTCXIO, and WTCX31.6 exponent {P = 0.001). Significantly different coefficient vs. Saline 
coefficient (P = 0.03). Significantly different coefficient vs. ROFAIOO coefficient {P = 0.0001). 


23 
































Table 10. Experiment A: Summary of Treatment-Related Histopathologic Findings in Mice One Day after 


Intratracheal Instillation of Particulate Matter Samples 


Treatment 

Group 

Bronchiole, 
Inflammation, 
Subacute, Focal 

Incidence Severity 

Bronchiole, 

Pigment, 

Free, Focal 

Incidence Severity 

Bronchiole, 

Pigment, 

Macrophage, Focal 
Incidence Severity 

Peribronchiolar, 
Inflammation, 
Acute, Focal 

Incidence Severity 

Saline 

0/8 

0.0 

0/8 

0.0 

0/8 

0.0 

0/8 

0.0 

MSH-100 

6/8 

0.8 

0/8 

0.0 

2/8 

0.3 

0/8 

0.0 

ROFA-10 

2/8 

0.3 

6/8 

0.8 

0/8 

0.0 

1/8 

0.1 

ROFA-100 

6/8 

1.0 

8/8 

1.5 

0/8 

0.0 

0/8 

0.0 

WTCX-10 

1/8 

0.1 

0/8 

0.0 

0/8 

0.0 

0/8 

0.0 

WTCX-31.6 

0/8 

0.0 

1/8 

0.1 

0/8 

0.0 

0/8 

0.0 

WTCX-100 

0/8 

0.0 

0/8 

0.0 

0/8 

0.0 

0/8 

0.0 


“ Incidence denotes number of mice in group with finding / total number of mice examined. Average severity score for 
the group is shown based on the following scoring system: 0 = not present, 1 = minimal, 2 = slight/mild, 3 = moderate, 
4 = moderately severe, 5 = severe/high. 


score (0.3) than the MSH-100 and ROFA-100 groups, and 
also had one mouse with minimal focal acute 
peribronchiolar inflammation. Although 1 mouse in the 






WTC-X group had a finding of minimal focal subacute 
bronchiolar inflammation, for an average group score of 
0 .1, this lesion was not found in any of the mice in the 



Figure 9. Experiment A: Representative micrographs of lesions occuring in lungs of mice one day after 
intratracheal instillation of PM samples or saline vehicle (all panels same magnification: bar length = 100 pm). 
A. Saline-instilled control mouse (#69) with no remarkable findings. B. Mouse #84 instilled with 100 pg 
MSH showing minimal degree of focal subacute bronchiolar inflammation. C. Mouse #57 instilled with 100 
pg pooled WTCX sample with no remarkable findings. D. Mouse #73 instilled with 100 pg ROFA showing 
slight/mild degree of focal subacute bronchiolar inflammation. 


24 












WTCX-31 .6 or WTCX-100 groups (Figure 9), suggesting 
that the lesion in the one WTCX-10 mouse was not 
treatment-related. Free bronchiolar pigment (presumably 
corresponding to PM) was identified in all ROFA-100 
mice at an average severity of 1.5 (Table 10), and in 6 of 
8 mice in the ROFA-10 group at an average severity of 
0.8. One mouse in the WTCX-31.6 group (but none in the 
WTCX-100 group) had minimal free bronchiolar pigment; 
again suggesting that this finding is not treatment- 
dependent. However, it may be more difficult to see the 
WTC PM which is lighter in color than the ROFA or MSH 
PM. Focal bronchiolar macrophage pigment was found in 
2 of 8 mice in the MSH-100 group at an average severity 
of 0.3. These findings indicate that both ROFA-100 and 
MSH-100, but not the pooled WTCX-100 or any lower 
dose, caused focal subacute bronchiolar inflammation. 

6. Summary. Results from investigation of the dose- 
response relationships of pooled WTCX PM showed that 
the two lower doses of WTCX (10 pg and 31.6 pg) did not 
have any significant effects on inflammatory parameters, 
lung histopathologic findings, or respiratory responses. 
The 100 pg dose of WTCX caused a slight but significant 
increase in BAL neutrophils (7% of total cells) as 
determined by BAL parameters, and no inflammation as 
determined by histopathologic examination, while the 
toxic PM control, ROFA, caused minimal inflammation by 
histopathologic examination, significant increases in BAL 
neutrophils and other cell types, and significant increases 
in biochemical indicators of lung injury. Despite the lack 
of effect of WTCX on lung injury and the relatively low 
level of neutrophilic inflammation, mice in the WTCX- 
100 group were significantly more responsive to Mch 
aerosol challenge than all other groups. A lack of 
correlation between lung inflammation and airway 
hyperresponsiveness is not uncommon (e.g. Alvarez et al., 
2000; Smith and McFadden Jr., 1995). The significant 
degree of airway hyperresponsiveness induced by WTC 
PMj 5 implies that components of the dust can promote 
mechanisms of airway obstruction. 

C. Experiment B: Effects of Nose-Only Inhalation 
Exposure 

1. Exposure results. The gravimetric concentration 
for the WTC3 exposure chamber was 10.64 ± 3.10 mg/m\ 
The mass median aerodynamic diameter (MMAD) was 
1.05 pm, and the geometric standard deviation (ag) was 
2.67. Chamber temperature and relative humidity was 74 
°F and 11% in the control chamber and 75 °F and 11% in 
the WTC3 chamber. The low humidity was required to 
prevent the PM from sticking to the string in the aerosol 
generation system; the humidity within the exposure tubes 


was significantly higher due to body heat from the mice in 
a confined environment. At the end of the exposure, two 
control mice (#131 and #146) and one WTC3-exposed 
mouse (#203) were found dead in the exposure tubes, 
apparently from attempting to turn around in the exposure 
tubes and suffocating. The incidence of this problem was 
not unusual considering the large number of mice exposed 
simultaneously (AD Ledbetter, personal communication). 
The two spare mice (designated #146a and #203a) were 
used to replace the dead ones, and were killed on day 6 
(December 3, 2001). An additional control mouse 
(designated #13la) was exposed to air for 5 hours on 
November 30, 2001 to replace the second dead control 
mouse, and was killed on day 3 (December 3, 2001). 
Therefore all groups of mice had the full number of 8 per 
group per time point. 


Table 11. Experiment B: Body Weights of Mice in Nose-Only 
Inhalation Exposure Study ® 


Day: 

Body Weight (e) 

-1 

0 

(Pre-) 

0.25 

(Post-) 

1 

3 

6 

Group 







Air 

23.91 

23.81 

21.83 

23.00 

24.04 

24.78 


0.16 

0.18 

0.18 

0.18 

0.23 

0.35 

WTC 3 

23.93 

23.47 

21.82 

23.05 

23.89 

24.66 


0.15 

0.18 

0.16 

0.18 

0.15 

0.34 


® Values shown are means (in bold) and SEM immediately below 
means (n=48 days -1 through 1, n=32 day 3, n = 16 day 6). Body 
weight was measured in the morning except on day 0.25 
(immediately after nose-only exposure). There was no significant 
difference between the two groups. 



Days after Exposure 


Figure 10. Experiment B: Body weights in nose-only inhalation 
exposure experiment. Values shown are means and SEM 
(numbers of mice shown in Table 11). Nose-only inhalation 
exposure caused a significant drop in body weight but there was 
no significant difference between groups. 


25 
















Table 12. Experiment B: Immediate Airway Responses “ 


Group 

Breathing Freauencv (min') 

PenH tunitless) 

Pre- 

Post- 

% increase 

Pre- 

Post- 

% increase 

Air 

549.2 

357.7 

-35.0 

0.88 

1.09 

29.5 


9.1 

24.4 

4.1 

0.05 

0.09 

13.8 

WTC 3 

560.3 

389.3 

-30.1 

0.94 

1.47 

60.1 


10.3 

19.0 

3.9 

0.06 

0.16 

18.2 


“ Values shown are means (in bold) and SEM immediately below means (n=12). Respiratory 
parameters were measured immediately before (Pre-) and after (Post-) nose-only inhalation 
exposure on day 0. No significant differences in percent change in frequency or PenH between 
groups were found. 


2. Body weights. Animal weights were monitored on 
days -1 (before exposure), 0 (both before and after 
exposure), 1, 3, and 6 (Table 11). Body weight was 
measured between 7:00 and 8:00 each morning, except 
immediately after exposure. There were no significant 
differences between the two groups at any time point. The 
nose-only exposure caused a significant 2 g drop in body 
weight in both groups of mice (Figure 10). 


3. Immediate airway responses to nose-only 
exposure. Ventilatory parameters were measured in 12 
mice from each group before and after the nose-only 
exposure. Ventilatory rate decreased after exposure in 
both groups but there was no significant difference 
between them (Table 12). It should be noted that many 
physiological responses are readily reversible, and the time 
required to unload the mice from the exposure tubes and 



Figure 11. Experiment B. PenH values measured immediately before and after nose-only exposure to WTC 3 PM or Air only. 
Legends refer to individual mouse numbers. Immediate response (calculated as [(Post-value - Pre-value) / Pre-value x 100%] was not 
significantly different between the two groups but data indicate the possibility of individual sensitivity to dust exposure. 


26 
































Table 13. Experiment B: Diffusing Capacity of the Lung for 
Carbon Monoxide “ 


Treatment 

Group 

Day after 

Treatment 

Subjects 

1 -4 

Subjects 

5-8 

Average 

n = 2 

Air 

1 

-3.761 

-4.185 

-3.973 

WTC 3 

1 

-3.715 

-3.981 

-3.848 

Air 

3 

-4.102 

-3.826 

-3.964 

WTC 3 

3 

-3.818 

-4.078 

-3.948 

Air 

6 

-3.528 

-4.162 

-3.845 

WTC 3 

6 

-3.487 

-3.809 

-3.648 


® Diffusing capacity of the lung for carbon monoxide was determined 
1, 3, or 6 days after exposure on four mice from each treatment 
group placed together in a single bell jar. Values shown are slopes 
of chamber [CO] vs. time (ppm/min), after subtraction of value from 
empty chamber. 

begin the measurement of breathing parameters (~20 
minutes) may have caused us to miss some changes. PenH 
was increased by an average of 30% after exposure to air 
and by an average of 60% after exposure to WTC3 (P 
0.20). Although this difference was not significant, 
examination of the changes in individual mice showed that 
PenH increased in all 12 mice exposed to WTC3, but only 
8 of 12 mice exposed to Air (Figure 11). Furthermore 
some of the increases in WTC3-exposed mice were quite 
large. These data indicate the possibility that individual 


mice in this outbred strain may be susceptible to 
bronchoconstrictive effects of WTC PM. 

4. DLCO measurements. DLCO was determined 1, 
3, and 6 days after exposure on 4 mice from each group 
placed together in the test chamber. Since there were 8 
mice per group per time point, only two tests of DLCO 
were conducted within each group, and no statistical 
comparison was possible between Air and WTC3 mice. 
Examination of the data showed little apparent difference 
in DLCO at different times in the two groups (Table 13). 

5. Responsiveness to methacholine aerosol. Analysis 
of baseline PenH values (immediately before Mch aerosol) 
between the two groups showed a significant difference 
depending on day, but not due to treatment (day 6 
baselines were lower in both groups; P = 0.0007; Table 
14). Responsiveness to increasing concentrations of Mch 
aerosol was assessed and quantified as described in the 
Methods section (Table 14). Unlike Experiment A, the 
results could be modeled with linear equations (Figure 12). 
Significant interactions of treatment, day, and Mch 
concentration were detected (P = 0.01), implying that the 
results depended upon a combination of factors. Slopes of 
the Day and Treatment combinations were significantly 
different (P = 0.0001). Analysis of the data showed that 
one equation could be used to describe the data for Air 
Day 6 and for WTC3 Day 1 (Figure 12). The slope of this 
line was significantly different from and less than that of 


Table 14. Experiment B: Baseline PenH and Responsiveness to Methacholine Aerosol “ 


Treatment 

Group 

Day after 

Treatment 

Baseline 

PenH 


Dose Mch (me/ml) and PenH AUC (PenH - sec) 

0 

4 

8 

16 

32 

64 

Air 

1 

0.80 

- 1.5 

20.0 

61.5 

262.0 

945.9 

1940.7 



0.04 

10.8 

15.3 

41.7 

71.4 

227.1 

597.3 

WTC 3 

1 

0.68 

21.8 

18.8 

88.9 

227.2 

697.8 

492.3 



0.05 

7.9 

20.8 

12.7 

50.0 

175.4 

104.0 

Air 

3 

0.76 

- 13.7 

50.4 

238.4 

384.9 

1188.1 

2031.4 



0.05 

9.8 

20.9 

68.7 

77.4 

172.5 

178.8 

WTC 3 

3 

0.86 

- 11.4 

15.2 

37.8 

198.6 

679.1 

1293.3 



0.07 

9.7 

8.3 

12.6 

108.8 

317.2 

356.6 

Air 

6 

0.61 

- 3.5 

- 7.6 

35.5 

106.5 

70.1 

786.6 



0.05 

3.8 

12.4 

11.7 

43.6 

59.5 

269.9 

WTC 3 

6 

0.58 

2.0 

31.1 

72.0 

276.7 

1030.4 

1935.6 



0.04 

2.9 

9.0 

20.4 

97.1 

252.8 

385.1 


“ Values shown are means (in bold) and SEM immediately below means (n=8 per group). Baseline PenH (enhanced 
pause; unitless) was measured immediately before methacholine aerosol challenge. "No significant differences were 
found between treatment groups, but there was a significant difference in day, with day 6 values being significantly 
lower than other days (solid line box; P = 0.0007). Methacholine aerosol (Mch) was administered (see Methods 
for details) at the indicated doses, and the airway response was calculated as the area under the curve (AUC) of 
the PenH response over time in seconds. See Figure 12 for description of statistical analysis of PenH AUC data. 


27 



















PenH Area Under Curve (PenH - sec) 


2500-1 


Air Day 1 


2000-1 

1500 

1000 - 

500 - 

0 


• Air Day 1 

PenH AUC = 30.53*[Mch]-122.6 


I f ' I ^ I I I I I I 't " i'i " i I I I I - i-] " r-r " i I I I I I I I I r 


2500 n 

2000 ^ 

1500 ^ 

1000 ^ 

500 - 


Air Day 3 


• Air Day 3 

PenH AUC = 30.53*[Mch]-122.6 




I I' I I I I I I I I I I I I I I I I I I I I I t I I I I I I I I I I I 


2500 i 

2000; 

1500 ; 

1000; 

500 - 


Air Day 6 


0 


• Air Day 6 ^ 

PenH AUC= 10.72*[Mch]- 17.76 


l-l I f I I I t I I I I' I I I I I I I I I I I I I I I I I I I 

0 10 20 30 40 50 60 70 


WTC3 Day 1 

• WTCDayl ^ 

PenH AUC= 10.72*[Mch]- 17.76 


I ? I' I I' 


i 


I 


I I I I I I 1 "f" 


I I I r 


I I I I I I I I I I I I I I i 1 


WTC3 Day 3 


WTCDay 3 

PenH AUC = 30.53*[Mch]-122.6 




I T I I i I » I T I > I I I I I I I I I I I I r I I I I I I I I I 


WTC3 Day 6 


WTC Day 6 

PenH AUC = 30.53*[Mch] - 122.6 


" I I I 1 I r I I T 


I I I T I I I I I I I I I I I I I I I I I I I I I I 


0 10 20 30 40 50 60 70 


[Mch] (mg/ml) 

Figure 12. Experiment B: Airway responsiveness to methacholine aerosol challenge in mice exposed nose-only to Air or aerosolized 
WTC (sample 3) and tested 1,3, or 6 days later (n = 8 /group; data shown are mean + SEM). Linear dose-response relationships were 
found. ® Slope of Air Day 6 and WTC3 Day 1 were significantly different from and lower than the 4 other groups. 


the equation used to fit the other four groups. It should be 
noted that as in Experiment A, once the lines were 
determined to come from groups with equal slopes, the 


28 


lines were fit simultaneously. This resulted in an equation 
for the common groups (e.g. Air Day 6 and WTC3 Day 1) 
that did not fit as well as lines fit to the individual group 






























Table 15. Experiment B: BAL Cell Numbers after Nose-Only Exposure ® 


Group 

Day 


BAL Cell Number (x 10"^) 




Mac 

Neut 

Eos 


Lym 

Air 

1 

14.80 

0.012 

0.003 


0.046 




3.11 

0.004 

0.002 


0.010 


WTC 3 

1 

17.48 

0.006 

0.000 


0.067 




3.13 

0.003 

0.000 


0.013 


Air ' 

3 

16.56 

0.008 

0.000 


0.125 




1.13 

0.004 

0.000 




WTC 3 

3 

26.72 

0.034 

0.016 

1 

0.197 

1 



3.11 

0.017 

0.009 

L_ 

0.036 

J 

Air 

6 

22.24 

0.000 

0.000 


0.140 




1.13 

0.000 

0.000 


0.026 


WTC 3 

6 

29.86 

0.019 

0.005 

1 

0.281 

i 



2.58 

0.008 

0.004 

l_ 

0.056 

j 


® Values shown are means (in bold) and SEM immediately below means (n=8 per 
group). Solid-line box: Significant difTerence(P=0.01)betweenAirandWTC3, 
Day 6 different from Day 1. Dashed-line boxes: Significant difference {P = 0.02) 
between Air and WTC3, Day 3 and Day 6 both different from Day 1. 


data. These results could be interpreted as saying that 
mice exposed to WTC3 became more responsive to Mch 
in the days following exposure, while Air-exposed mice 


became less responsive. However, close 
examination of the data from experiment B 
showed it was more variable than that from 
experiment A. Therefore, although the Air Day 6 
and WTC3 Day 1 groups were less responsive to 
Mch aerosol challenge than the other four groups, 
the biological significance of this finding is 
unclear. 

6. BAL parameters. Numbers of BAL cells 
were quantified 1, 3, and 6 days after nose-only 
exposure to Air or WTC3 (Table 15). Analysis of 
the data showed that mice exposed to WTC3 had 
significantly greater numbers of macrophages (P 
= 0 . 01 ) and lymphocytes (P = 0 . 02 ) compared 
with Air-exposed mice. Macrophage numbers 
were significantly greater on Day 6 vs. Day 1, and 
lymphocyte numbers were significantly greater on 
both Day 3 and Day 6 vs. Day 1. Over all time 
points, WTC3 mice had 38% more macrophages, 
and 75% more lymphocytes (Figure 13). 
However, macrophages comprised 99% of all recovered 
cells in both groups at all time points. Lymphocytes 
constituted about 1 % or less of total BAL cells, while both 



Day 1 Day 3 Day 6 Day 1 Day 3 Day 6 


Figure 13. Experiment B: Bronchoalveolar lavage cell numbers recovered from mice 1, 3, or 6 days after 5 hr 
nose-only inhalation exposure to WTC sample 3 or Air only. Values shown are means and SEM (n=8 per group). 
Cell types shown are macrophages and monocytes (Mac), eosinophils (Eos), neutrophils (Neut), and lymphocytes 
(Lym). “ Significant difference (P = 0.01) between Air and WTC3, Day 6 different from Day 1. ^ Significant 
difference (P = 0.02) between Air and WTC3, Day 3 and Day 6 different from Day 1. 


29 




















































Table 16. Experiment B: BAL Supernatant Biochemical Values after 
Nose-Only Exposure “ 


Group 

Day 

Protein 

pg/ml 

Albumin 

pg/ml 

LDH 

U/L 

NAG 

U/L 

Air 

1 

165.2 

21.0 

29.0 

1.5 



6.1 

1.1 

3.4 

0.1 

WTC3 

1 

147.1 

16.9 

23.9 

1.6 



6.7 

1.2 

3.3 

0.1 

Air 

3 

136.6 

16.2 

33.0 

1.8 



10.4 

1.2 

6.4 

0.0 

WTC3 

3 

138.1 

15.8 

28.9 

1.6 



7.8 

1.5 

3.4 

0.1 

Air 

6 

172.6 

22.4 

30.2 

1.4 



8.5 

1.3 

2.4 

0.2 

WTC3 

6 

146.5 

17.5 

27.1 

1.4 



6.8 

1.1 

3.1 

0.1 


® Values shown are means (in bold) and SEM immediately below means (n=8 per 
group). Heavy solid-line boxes: Significant overall treatment effect (WTC3 < Air; 
no significant day effect); P = 0.05 (Protein) or P = 0.007 (Albumin). 


neutrophils and eosinophils were about 0 . 1 % or less of 
total BAL cells, indicating that WTC3 did not induce a 
significant acute inflammatory reaction. The increase in 


macrophages and lymphocytes is probably a 
nonspecific reaction to inhalation of large 
amounts of dust which induces macrophage 
recruitment for phagocytosis and clearance of 
the particles (Adamson and Bowden, 1981). 

Levels of proteins and enzymes in the BAL 
supernatant were assessed in the two groups of 
mice at 3 time points (Table 16). Significant 
differences between Air and WTC3 groups were 
found for total protein {P = 0.05) and albumin {P 
= 0.007), but there was no significant day effect. 
Surprisingly, the levels of protein and albumin 
were higher in the Air group. However, the 
overall levels of all proteins and enzymes was 
low in both groups and at all time points (Figure 
14), in comparison with Experiment A. The 
results indicate that at this exposure 
concentration and duration, WTC3 PM 2 5 does 
not induce severe acute lung injury. 

7. Nasal histopaihology. No exposure- 
related nasal lesions were found in mice exposed 
to air alone (controls). Similarly no nasal lesions were 
found in the mice exposed to WTC3 and killed 3 or 6 days 
post-exposure. None of the mice in any group had 


Protein 



Day 1 Day 3 Day 6 


75-1 


J 50- 



Day 1 


LDH 


[ZZIAir 

■■iWTC3 


T 



L 


1 


Day 3 Day 6 


Albumin 



CZDAir 

^WTC3 


a 



Day 1 Day 3 Day 6 


NAG 



Figure 14. Experiment B: BAL supernatant biochemical values in mice 1, 3, or 6 days after 5 hr nose-only 
inhalation exposure to WTC3 or Air only. Values shown are means and SEM (n=8 per group). ® Significant overall 
treatment effect (WTC3 < Air; no significant day effect); P = 0.05 (Protein) or P = 0.007 (Albumin). 


30 

















































































exposure-related alterations in the mucosal tissues lined by 
respiratory or olfactory epithelium in the more distal tissue 
sections examined (T2 and T3). 

The only nasal alterations observed by light 
microscopic examination was minimal to mild acute, focal 
inflammation (rhinitis) in four of the eight mice exposed 
to WTC3 PM 2 5 and killed 24 h post-exposure (animal # 
161,162,163,164). This minimal inflammatory response 
was bilateral and restricted to the most proximal tissue 
section examined (Tl). It was characterized by a slight 
increase in the number of neutrophils in the mucosal 
tissues lining the lateral meatus, especially in the ventral 
lateral meatus, the dorsomedial aspect of the proximal 
maxilloturbinate, and the ventral aspect of the proximal 
nasoturbinate in both nasal passages. It must be 
emphasized, however, that the severity of this focal rhinitis 
was minimal to mild (i.e., severity score of 1 or 2 out of 4). 
In addition, there were no associated histologic alterations 
in the surface epithelium or in the subepithelial tissues in 
the affected areas. In mouse # 162 there was a small 
accumulation of mucus and fiber-like material in the 
lateral meatus of one nasal passage in Tl. 

In summary, some but not all mice exposed to WTC3 
and killed 1 day after exposure had a minimal acute 
rhinitis that was restricted to the proximal nasal airways. 
This minimal inflammatory response was probably due to 
stimulation by the WTC3 exposure. This stimulation, 
however, did not result in any apparent epithelial cell 
injury that is often observed with many inhaled agents. No 
nasal lesions were observed in mice exposed to WTC3 and 
killed 3 or 6 days post-exposure. This suggests that any 
acute inflammation that may have been induced by the 
dust exposure quickly resolved and did not result in any 
persistent injury to the nasal mucosa that could be detected 
by light microscopy. 

8. Lung histopathology. No remarkable findings 
were observed in any of the mice exposed to Air or to 
WTC3 at any time point. Since nasal lesions as described 
above were restricted to the proximal Tl region and were 
not found in the more distal T2 and T3 regions, the lack of 
any findings in the lung suggests that the proximal region 
of the nose effectively scrubbed out enough of the 
particulate matter during the exposure to WTC3 to limit 
deposition further down the respiratory tract. It should be 
noted that mice are obligate nose-breathers, while humans 
have significant oral breathing, and therefore significantly 
more PM can bypass the nasal passages in humans 
(Schlesinger, 1985). Studies have shown considerably less 
deposition efficiency in the alveolar region of rodents 
compared with humans (Asgharian et al., 1995). 


9. Summary. Results from investigation of the 
effects of nose-only exposure to the WTC3 sample 
indicate that WTC3 PM 25 induced mild transitory 
neutrophilic inflammation in proximal nasal airways of 
some mice, but WTC3 PMj 5 did not induce neutrophilic 
inflammation in the lungs of any mice. However, numbers 
of macrophages were significantly increased after 
exposure, suggesting that some WTC3 PM 2 5 penetrated 
into the lower respiratory tract, which stimulated 
recruitment of macrophages to phagocytize and clear the 
particulate matter. Biochemical parameters of lung injury 
were not increased at all by WTC3. The data suggested 
that individual mice in this outbred strain may be sensitive 
to the immediate effects of WTC3 exposure and respond 
with increased airway obstruction, although this effect was 
not significant for the group as a whole. Groups of mice 
exposed to Air or WTC3 PM 2.5 differed in their 
responsiveness to Mch aerosol at different times after 
exposure, but the biological significance of these results 
was unclear. The dose deposited in the respiratory tract 
following nose-only inhalation may be estimated as 
follows: 18.8 ml/min (mouse minute ventilation based on 
weight; Costa et al., 1992) x 300 min (exposure time) x 
0.001 L/ml X 0.001 vc?fL x 10.64 mg/m^ (exposure 
concentration) x 1000 pg/mg x 0.23 (deposition efficiency 
estimate in total respiratory tract) == 14 pg. Thus, the 
significant difference in dose deposited into the airways 
between oropharyngeal aspiration (100 pg) and nose-only 
inhalation probably accounts for the lack of effect in many 
of the endpoints examined following nose-only inhalation 
exposure. 

D. Experiment C: Effect of Geographical Location of 
WTC PM Samples on Responses 

1. Sub-experiments and body weights. WTC PM 2 5 
samples from 7 different sites comprised the pooled 
WTCX sample (Figure 1, Table 1). The effects of the 
pooled sample may have been dominated by one or more 
site samples which were toxic in comparison with other 
site samples. Experiment C was designed to address this 
possibility and to examine the variability of pulmonary 
responses associated with WTC PM 2 5 samples collected 
from different geographical locations. The 7 sites were 
located east (WTC 11 - 0.1 miles, WTC 8 - 0.4 miles), 
southeast (WTC 13 - 0.1 miles, WTCF - 0.25 miles), south 
(WTCB - 0.25 miles), west-northwest (WTCC - 0.2 miles), 
and north-northeast (WTCE - 0.25 miles) from the center 
point of Ground Zero. Sub-experiment Cl examined 
responses to WTC 8 , WTC 13, WTCF, NIST, and Saline 
control mice. Sub-experiment C2 examined responses to 
WTC 11, WTCB, WTCC, WTCE, and Saline control mice. 


31 









Table 17. Experiment C: Body Weights “ 


Group 

Sub- 


Body Weight (e) 


Experiment 

Day -1 

Day 0 

Day 1 

Day 3 

Saline 

Cl 

24.69 

24.76 

24.72 

24.91 



0.39 

0.42 

0.45 

0.42 

WTC8 

Cl 

24.76 

24.54 

24.16 

25.35 



0.49 

0.52 

0.58 

0.90 

WTC 13 

Cl 

24.69 

24.26 

23.91 

24.14 



0.40 

0.42 

0.38 

0.43 

WTCF 

Cl 

24.72 

24.46 

24.17 

24.41 



0.43 

0.42 

0.38 

0.49 

NIST 

Cl 

24.75 

24.32 

24.15 

24.31 



0.50 

0.53 

0.50 

0.71 

Saline 

C2 

nd'’ 

22.55 

22.23 

22.85 




0.27 

0.25 

0.55 

WTC 11 

C2 

nd 

23.72 

23.20 

23.72 




0.39 

0.43 

0.68 

WTCB 

C2 

nd 

23.66 

23.11 

23.29 




0.33 

0.33 

0.61 

WTCC 

C2 

nd 

23.67 

23.12 

23.96 




0.27 

0.24 

0.41 

WTCE 

C2 

nd 

23.69 

23.27 

23.44 




0.29 

0.36 

0.41 


* Values shown are means (in bold) and SEM immediately below means (on days -1,0, and 
1, n=l 6 per group, except Saline sub-experiment C2; n=8; on day 3, n=8 per group, except 
Saline sub-experiment C2: n=4). ** nd - Not determined. No treatment-related differences 
in body weight among groups within each sub-experiment were detected. 


Responses were examined in 8 mice per group at 1 and 3 
days after oropharyngeal aspiration of 100 |ag of each PM 
sample or saline alone (n = 4 per time point in sub¬ 
experiment C2 Saline mice). Responses were examined at 
both 1 and 3 day time points in order to begin examination 
of persistence of exposure effects. Statistical analysis of 
the data was performed within each sub-experiment. 

Body weights were determined on days -1 (before 
oropharyngeal aspiration), 0, 1, and 3 in sub-experiment 
Cl, and on days 0, 1, and 3 in sub-experiment C2 (Table 
17). No treatment-related differences in body weight 
among groups within each sub-experiment were detected, 
although there were differences on the day the animals 
were weighed {P = 0.0001). 

2. Responsiveness to methacholine aerosol. In sub¬ 
experiment C1, the WTC8 group had significantly greater 
baseline PenH values 1 day after exposure compared with 
the WTC13 group (Table 18). No other significant 
differences in baseline PenH values in sub-experiments C1 


or C2 were found. Responsiveness to methacholine 
aerosol was quantified as PenH AUC (Table 18). Analysis 
of the data in both sub-experiments showed that linear 
regression equations could be fit to the PenH AUC vs. 
[Mch] data (Figure 15). In both sub-experiments, tests for 
equal slopes on days 1 and 3 after exposure showed that 
day was not a significant factor. Therefore, a single 
equation was fit to the data for each group, and day does 
not appear in the equations. 

In sub-experiment Cl, the WTC8, WTCF, and NIST 
groups could be described with a common slope and 
intercept. The common slope of these 3 groups was 
significantly different from and greater than that of the 
WTC13 or Saline Cl groups (P< 0.0005), indicating that 
WTC8, WTCF, and NIST were hyperresponsive to 
methacholine aerosol. The slope of the WTC13 group was 
significantly greater than that of the Saline Cl group, 
showing that WTC 13 mice were hyperresponsive 
compared with control mice, though less so than WTC8, 


32 










Table 18. Experiment C: Baseline PenH and Responsiveness to Methacholine Aerosol 


Group 

Sub- 

Experiment 

Day 

Baseline 

PenH 


Dose Mch (me/ml) and PenH AUC (PenH - sec) 


0 

4 

8 

16 

32 

64 

WTC8 

Cl 

1 

0.68 

35.2 

69.6 

197.0 

429.4 

1115.0 

1953.5 




0.05 

10.1 

19.2 

92.8 

87.0 

126.6 

203.5 

WTC 13 

Cl 

1 

0.45 

35.5 

50.5 

87.9 

264.7 

674.8 

1151.3 




0.02 

5.1 

4.4 

9.1 

44.2 

135.4 

206.6 

WTCF 

Cl 

1 

0.55 

25.9 

62.3 

164.4 

369.1 

1145.3 

1966.5 




0.05 

2.1 

14.4 

29.0 

47.3 

155.8 

293.0 

NIST 

Cl 

1 

0.59 

31.6 

52.7 

97.3 

279.3 

811.1 

1882.2 




0.03 

3.1 

2.9 

11.5 

54.0 

149.2 

324.6 

Saline 

Cl 

1 

0.57 

24.2 

40.4 

61.4 

171.7 

534.5 

1077.0 




0.02 

1.5 

4.0 

6.6 

19.9 

74.2 

163.2 

WTC8 

Cl 

3 

0.66 

14.5 

41.9 

62.2 

182.5 

690.3 

1907.0 




0.04 

3.0 

10.9 

25.1 

49.7 

206.4 

370.0 

WTC 13 

Cl 

3 

0.78 

27.9 

51.2 

207.0 

271.9 

1000.8 

1554.5 




0.08 

9.0 

9.7 

62.6 

68.3 

204.3 

190.8 

WTCF 

Cl 

3 

0.77 

18.8 

29.3 

87.5 

280.8 

994.4 

2160.8 




0.07 

6.4 

22.3 

34.4 

130.7 

368.1 

563.2 

NIST 

Cl 

3 

0.76 

21.1 

47.1 

86.1 

212.3 

962.5 

1886.2 




0.05 

8.0 

14.8 

19.4 

72.2 

271.0 

437.2 

Saline 

Cl 

3 

0.71 

- 2.8 

30.6 

99.2 

280.4 

481.0 

1287.9 




0.03 

4.6 

17.2 

16.8 

67.0 

40.8 

79.5 

WTC 11 

C2 

1 

0.69 

15.9 

66.9 

238.8 

811.0 

1496.5 

1835.9 




0.06 

6.5 

23.3 

90.7 

223.6 

437.7 

306.0 

WTCB 

C2 

1 

0.67 

7.3 

46.4 

205.4 

695.5 

1561.4 

2333.1 




0.05 

9.4 

10.0 

53.8 

193.0 

404.0 

611.8 

WTCC 

C2 

1 

0.62 

5.9 

27.5 

118.7 

737.6 

1826.9 

2800.2 




0.06 

5.9 

9.9 

30.6 

249.0 

466.3 

672.8 

WTCE 

C2 

1 

0.62 

8.3 

28.7 

155.6 

390.0 

1112.5 

1722.8 




0.02 

3.1 

12.6 

86.8 

177.1 

360.2 

452.6 

Saline 

C2 

1 

0.61 

3.2 

52.5 

111.7 

167.7 

308.7 

825.5 




0.08 

4.0 

7.5 

19.3 

29.5 

26.0 

101.6 

WTC 11 

C2 

3 

0.59 

3.8 

24.7 

45.0 

256.8 

1003.0 

2304.1 




0.05 

6.3 

12.5 

19.2 

112.0 

325.5 

559.8 

WTCB 

C2 

3 

0.59 

22.2 

31.9 

98.0 

238.6 

738.5 

2609.2 




0.05 

4.3 

14.6 

34.7 

59.2 

216.6 

585.5 

WTCC 

C2 

3 

0.56 

10.0 

22.8 

118.9 

351.7 

1282.4 

2669.0 




0.04 

7.1 

6.6 

37.4 

144.1 

365.5 

520.9 

WTCE 

C2 

3 

0.62 

5.7 

42.3 

138.6 

412.4 

1257.9 

2088.7 




0.05 

6.3 

11.7 

33.2 

117.6 

297.5 

472.9 

Saline 

C2 

3 

0.62 

3.5 

27.5 

84.0 

114.7 

341.1 

1058.7 




0.05 

3.5 

27.0 

65.1 

17.2 

65.2 

45.7 


“ Values shown are means (in bold) and SEM immediately below means (n=8 per group, except Saline sub-experiment 
C2: n=4). A significant difference in baseline PenH (enhanced pause; unitless) was found between the WTC8 group 
and the WTC13 group on day 1 (heavy solid line box). No other significant differences in baseline values on day 1 or 
day 3 were detected. Methacholine aerosol (Mch) was administered at the indicated doses, and the airway response was 
calculated as the area under the curve (AUC) of the PenH resjxinse over time in seconds. See Figure 15 for description 
of statistical analysis of PenH AUC data. 


33 






























PenH Area Under Curve (PenH - sec) 


3000- 


2000 - 


1000 - 


0 -<>- 


WTC8 

a 


PenH AUC = 32.8*[Mch} - 139.4 
• 8 Day 1 

o 8 Day 3 


2 


i 

* o 

( % I I—I—I—I—I—I—I—I—I—I—I—r 


I I I I I I I —n— I 't 'T' t -i——I 


WTC 11 

c 

PenH AUC = 38.5* [Mch] - 103.9 
• 11 Day 1 

O 11 Day 3 

I 


o 

A 


I 

« 2 




3000- 


2000 - 


1000 - 


WTCF 

a 


-PenH AUC = 32.8*[Mch] - 139.4 

• F Day 1 

O F Day 3 


I 


0-<h-#-r¥ 


I I T I r I I ■ I T - T -y" I i i i { •! “i r‘ T ‘'|—r'T'VT-] 


I I I'’!- I [ I I I r I I 1 I—r 1 I I I—I I I I I I I > I I 

WTCB 

c 

- PenH AUC = 38.5* [Mch] - 103.9 
B Day 1 


O B Day 3 




I 

8 ^ 


I 


2 


NIST 1649a 



1^1 I I I I I I f r T ' T ‘ V ‘] "r -T--i I [ r - r I i 1 7 T - r - r - v ] 

WTCC 

c 

Pen H A UC = 38.5* [Mch] - 103.9 
CDay 1 


O C Day 3 

I 

s 


I 

1 


r- TM - T -i I I I I' I' I " I "I 


WTC E , Saline C2 

c 

PenH AUC = 38.5*[Mch]- 103.9 
• EDay 1 
O E Day 3 

PenHAUC= 14.9*[Mch] -61.1 
A Saline C2 Day 1 
A Saline C2 Day 3 o 


I 


I 

I 

A 




; I I I t I I I I I I I I > I } I I I 1 } I ’I r r I 1 

10 20 30 40 50 60 70 0 10 20 30 40 50 60 


-I—i—i—] 

70 


[Mch] (mg/ml) 

Figure 15. Experiment C: Airway responsiveness. See next page for figure legend. 


34 

















































Figure 15. (previous page.) Experiment C: Airway 
responsiveness to methacholine aerosol challenge in mice 
exposed to saline vehicle, NIST 1649a, or WTC PM samples 
from individual collection sites and tested 1 or 3 days later (n 
= 8 per group except Saline sub-experiment C2: n = 4). A 
single regression equation was fit to the data for both days in 
each group. “ In sub-experiment Cl (left panels), a common 
equation could be fit to the WTC8, WTCF, and NIST data, 
and the slope of the line was significantly different from and 
greater than the slopes of the WTC 13 and Saline Cl 
equations. ^ In sub-experiment Cl (left panels), the slope of 
the equation for the WTC 13 group was significantly different 
from and greater than the slope for the Saline C1 group. In 
sub-experiment C2 (right panels), a common equation could 
be fit to the WTCl 1, WTCB, WTCC, and WTCE data, and 
the slope of the line was significantly different from and 
greater than the slope of the Saline C2 equation. 


WTCF, and NIST mice. 

In sub-experiment C2, the WTCl 1, WTCB, WTCC, 
and WTCE groups could all be described with a common 
slope and intercept, which was similar to that found for 
WTC8, WTCF, and NIST groups in sub-experiment Cl. 
The common slope of the 4 WTC groups was significantly 
different from and greater than that of the Saline C2 group 
{P = 0.001), indicating that WTC 11, WTCB, WTCC, and 
WTCE were hyperresponsive to methacholine aerosol. 

In general, these results are consistent with those from 
Experiment A, where the 100 pg dose of the pooled 
WTCX sample induced significant hyperresponsiveness to 
methacholine aerosol compared with control PM samples 
and saline. All but one of the WTC PM samples, as well 
as the NIST control PM, appeared to cause similar degrees 
of hyperresponsiveness. However, the WTC 13 sample, 
located just 0.1 miles southeast of Ground Zero, caused a 
lower degree of hyperresponsiveness compared with 
WTC8, WTCF, and NIST. 

3. BAL cells. After assessment of responsiveness to 
Mch aerosol, mice were killed and numbers of BAL cells 
were quantified (Table 19; Figure 16). In sub-experiment 
C1, significant increases in numbers of neutrophils on Day 
1 were found in all PM-exposed groups compared with 
Saline C1 mice. An average of 14.7x10^ neutrophils was 
recovered from NIST mice (45% of total BAL cells). 
Significantly lower numbers of neutrophils were found in 
WTC 13 (6.1 X 10^) and WTCF (6.9 x 10") mice, while 
numbers of neutrophils were lower still in WTC8 mice 
(3.2 X 10"). The neutrophilic response abated by Day 3, 
and there were no significant differences among the 5 
groups. Numbers of lymphocytes were significantly 
increased in WTC8, WTC 13, WTCF, and NIST mice in 


comparison with Saline C1 mice on both Day 1 and Day 
3 after oropharyngeal aspiration {P = 0.0001). 
Lymphocyte numbers significantly increased in all groups 
from Day 1 to Day 3 (P = 0.0001). Since there were 
significant interactions between day and treatment with 
respect to eosinophil numbers {P = 0.01), no significant 
differences among groups could be discerned. Although 
a significant difference in macrophage numbers was 
detected in the WTC 13 group compared with saline, it was 
very marginal and not considered biologically significant. 

In sub-experiment C2, significant increases in 
neutrophils and eosinophils were found in WTCl 1 and 
WTCE mice compared with Saline C2 mice. The average 
number of neutrophils in these 2 WTC groups was 
comparable to those found in the WTC 13 and WTCF 
groups in sub-experiment Cl. In addition, numbers of 
neutrophils and eosinophils were significantly greater in 
WTCE mice compared with WTCB mice. Neutrophils 
numbers declined from Day 1 to Day 3 (P = 0.0001). It 
should be noted that of the four mice in the Saline C2 Day 
1 group, two had unusually high neutrophil numbers 
(individual numbers: 0.15, 0.26, 3.15, and 5.79 x 10" 
neutrophils), which limited the ability to determine 
significant increases in neutrophils in mice exposed to 
WTC PM samples in sub-experiment C2. The reasons for 
this significant inflammatory response in two control mice 
are not apparent, but this finding does not detract from the 
overall conclusion that PM collected from specific 
locations near the WTC site caused significant 
inflammation of neutrophils and eosinophils. 

These results differ substantially from those found in 
Experiment A, where 100 pg of pooled WTCX induced 
only a mild neutrophilic response in the lung one day after 
oropharyngeal aspiration (average 1.43 x 10"). Some 
WTC individual site samples (WTCF, WTC 13, WTCl 1, 
WTCE) caused about 4 times the amount of neutrophil 
recruitment as WTCX, while the others (WTC8, WTCB, 
WTCC) caused about twice as much recruitment. It is not 
clear how the individual site samples could all cause more 
lung inflammation than the pooled WTCX sample which 
was composed of the individual site samples. This finding 
may be a result of significant differences in responsiveness 
of different lots of mice sent on different weeks, which we 
have found with some studies of other toxic inhalants. 
Additionally, although these mice were lavaged after Mch 
challenge, our experience has shown that the challenge 
itself does not induce cellular inflammation that might 
account for the observations made here. To adequately 
address this question, pooled WTCX and individual site 
samples would need to be tested together in the same 
experiment. 


35 













Table 19. Experiment C: BAL Cell Numbers ® 


Group 

Sub- 

Experiment 

Dav 

Mac 

BAL Cell Number ( 

Neut 

X 10-") 

Eos 


Lvm 

Saline 

Cl 

1 

16.41 

0.34 



0.02 


0.14 




1.92 

0.20 



0.01 


0.02 

WTC8 

Cl 

1 

21.43 

3.23 



0.27 


0.38 




2.38 

0.52 



0.08 


0.05 

WTC13 

Cl 

1 

24.19 

6.10 



1.33 


0.70 




2.24 

0.88 



0.43 


0.14 

WTCF 

Cl 

1 

18.47 

6.85 



0.84 


0.58 




1.45 

0.85 



0.22 


0.10 

NIST 

Cl 

1 

17.36 

14.67 



0.43 


0.48 




3.41 

1.17 



0.17 


0.13 

Saline 

Cl 

3 

24.03 

0.48 



0.19 


0.36 




6.56 

0.45 



0.07 


0.09 

WTC8 

Cl 

3 

29.79 

0.18 



1.48 


0.75 




2.70 

0.06 



0.90 


0.15 

WTC13 

Cl 

3 

25.96 

0.46 



0.39 


0.88 




2.32 

0.15 



0.10 


0.22 

WTCF 

Cl 

3 

24.60 

0.25 



1.46 


1.16 




2.23 

0.07 



0.49 


0.26 

NIST 

Cl 

3 

29.09 

1.74 



0.67 


1.97 




4.06 

0.26 



0.36 


0.41 

Saline 

C2 

1 

16.25 

2.34 



0.22 


0.16 




1.21 

1.34 



_0_H9_ 


0.06 

WTCll 

C2 

I 

23.38 

5.79 

1 

0.75 

1 

0.98 




3.84 1_M2_ 

_L_ 


J 

0.26 

WTCB 

C2 

1 

25.91 

2.98 



0.17 


0.49 




3.90 

0.95 



0.05 


0.05 

WTCC 

C2 

1 

27.19 

2.53 



0.28 


0.29 




4.02 

0.42 



0.10 


0.08 

WTCE 

C2 

1 

24.19 

5.12 



0.37 


0.38 




2.94 

0.80 



0.12 


0.10 

Saline 

C2 

3 

20.87 

0.03 



0.02 


0.27 




0.69 

0.02 



_0_^1_ 


0.11 

WTCll 

C2 

3 

25.50 

1.13 

1 

0.49 

1 

0.75 




3.80 1 _0^9_ 

_ 1 __ 

_0J.6_ 

J 

0.31 

WTCB 

C2 

3 

29.60 

0.16 



0.30 


0.46 




2.87 

0.05 



0.12 


0.11 

WTCC 

C2 

3 

24.76 

0.34 



0.39 


0.79 




2.24 

0.08 



0.14 


0.19 

WTCE 

C2 

3 

30.11 

0.21 



1.59 


1.45 




2.62 

0.05 



0.43 


0.50 


“ Values shown are means (in bold) and SEM immediately below means (n=8 per group, except Saline sub-experiment 
C2: n=4). Significant differences shown are within sub-experiments only. Heavy solid-line boxes: Within 
sub-experiment Cl day 1 neutrophils, NIST > WTC13 and WTCF > WTC8 > Saline. Solid-line boxes, underlined 
values: NIST, WTC13, WTCF, and WTC8 all significantly different from Saline. Dashed-line boxes: WTCll 
significantly different from Saline. Solid-line shaded boxes: WTCE significantly different from Saline and WTCB. 


36 





































Cells (10^) Cells (lO'*) Cells (10 ) Cells (10 ) 


40-, 


30- 


20 - 


10 - 


3 Saline (Cl) 
3 WTC8 
WTC13 


ESS23 WTCF 
^■UNIST 


X 


X 


Day 1 


20-1 


15- 


10 - 


5-1 


Day 1 


I I Saline (Cl) 


cnzzo WTC8 
WTC13 
issssa WTCF 
■■■NIST 


Mac 


-r J 



Day 3 


Neut 



C=] Saline (Cl) 
EIZZ3 WTC8 
WTC13 
WTCF 
■■■NIST 


Day 3 


Eos 


jiXl 


T 




5-1 


Day 1 


Saline (Cl) 

WTC8 

WTC13 


Day 3 


Lym 


WTCF 



40-1 


30- 


vzzm WTCB 
iTTrn WTCC 
. ISSS3 WTCE 


o 


^ 20 - 

u 

10-1 


3 Saline (C2) 
WTCll 


Mac 


X 


1 






1 


1^ 


1 




I 


I 




Day 1 


Day 3 




Day 1 


Day 3 



Day 1 Day 3 

Figure 16. Experiment C: BAL cells. See next page for figure legend. 


37 















































































































































































































































































































































Figure 16. (previous page.) Experiment C: BAL cell 
numbers recovered from mice exposed to saline vehicle, NIST 
1649a, or WTC PM samples from individual collection sites 
and tested 1 or 3 days later (n = 8 per group except Saline 
sub-experiment C2: n = 4). Cell types shown are 
macrophages (Mac), neutrophils (Neut), eosinophils (Eos), 
and lymphocytes (Lym). “ NIST significantly greater than all 
other groups. ^ WTC 13 and WTCF significantly greater than 
WTC8 and Saline Cl groups. WTC8 significantly greater 
than Saline Cl group. ^ Lymphocyte numbers significantly 
greater in WTC8, WTC 13, WTCF, and NIST groups 
compared with Saline Cl group. ® Significantly greater 
numbers of neutrophils and eosinophils in WTC 11 group 
compared with Saline C2 group. ‘ Significantly greater 
numbers of neutrophils and eosinophils in WTCE group vs. 
WTCB and Saline C2 groups. 


4. BAL proteins and enzymes. As for other 
parameters, BAL protein and enzyme data for sub¬ 
experiments Cl and C2 were analyzed separately (Table 
20, Figure 17). In sub-experiment Cl, significant 
increases in BAL total protein levels were found in the 
NIST group compared with the WTC8 group {P = 0.05). 
No significant differences due to Treatment were found 
with respect to albumin or LDH levels. There were 
significant interactions between Day and Treatment in 
NAG values (P = 0.02), indicating the results depended on 
the day animals were killed. 

In sub-experiment C2, there were significant effects of 
Day after treatment for total protein and LDH, but there 
were no effects of Treatment group. There were 
significant interactions between Day and Treatment in 
NAG values {P = 0.005), indicating the results depended 
on the day after treatment. 

In both sub-experiment Cl and C2, one of the saline 
group mice killed on Day 1 had very high values for total 
protein, albumin, and LDH, which increased the mean 
values and variability in these groups. Although this result 
may have limited the ability to detect some statistical 
differences, overall the biochemical values were not 
greatly different among the treatment groups, and any 
additional differences with more consistent control data 
would likely have been minimal. Therefore the results for 
the individual site WTC PM samples are comparable to 
those found with the pooled WTCX sample in Experiment 
A, where no differences from control saline mice were 
found. 

5. Lung histopathology. Following tests for airway 
responsiveness to Mch aerosol and lung lavage, lungs were 
removed and fixed with 4% paraformaldehyde, and 


pathological changes were assessed. Although the lungs 
of all mice in Experiment C were lavaged (they were not 
lavaged in Experiments A or B), the pattern and the 
morphology of the PM induced findings were relatively 
consistent in all treated groups. 

Focal subacute bronchiolar inflammation and focal 
bronchiolar pigmented macrophages (presumably PM) 
were consistently observed in all groups of mice dosed 
with each of the different PM samples, and both findings 
are considered to be PM-induced in all groups (Table 21). 
Some groups also had findings of focal free bronchiolar 
pigment, consistent with the pigment in macrophages. No 
remarkable findings were observed in the lungs of the 
saline control group (Figure 18A), except for one mouse 
which had a minimal degree of focal subacute bronchiolar 
inflammation which was not considered to be treatment- 
related. Table 21 shows the rankings of the treatment- 
related histopathologic findings in mice 1 or 3 days after 
exposure. The degree of focal subacute bronchiolar 
inflammation was greatest in the NIST (Figure 18C), 

WTCE, and WTC 13 (Figure 18D) groups on Day 1 
(average severity scores of 1.9,2.0, and 2.1, respectively). 
The scores in the WTCC (Figure 18B), WTCB, WTC8, 

WTCF, and WTCl 1 groups were lower (average severity 
scores of 0.8, 1.1, 1.1, 1.3, and 1.3, respectively). By Day 
3, the focal subacute bronchiolar inflammation was 
greatest in the NIST group (average severity score 2.1; 
Figure 18E), while the scores were reduced in all of the 
WTC PM groups relative to their scores on Day 1 (Figure 
18F). 

The histopathologic scoring system is semi- 
quantitative, and much larger numbers of mice per group 
would be necessary to determine statistically significant 
differences among groups. Nevertheless, these results also 
show substantial differences from those found in 
Experiment A with the pooled WTCX sample. 
Oropharyngeal aspiration of 100 pg of WTCX did not 
cause any treatment-related histopathologic findings. In 
contrast, all individual site samples of WTC PM induced 
at least minimal focal subacute bronchiolar inflammation, 
and some samples caused slight/mild and even moderate 
degrees of inflammation. In addition, pigment associated 
with PM was visible in macrophages from all WTC PM- 
exposed mice, but none was visible in mice exposed to 
pooled WTCX in Experiment A. Re-examination of the 
slides by a different observer will be necessary to confirm 
this finding. The findings of pulmonary inflammation in 
WTC PM groups by histopathologic examination are 
consistent with the results from the quantification of BAL 
cell numbers. 


38 













Table 20. Experiment C: BAL Supernatant Biochemical Values “ 



Sub- 


Protein 

Albumin 

LDH 

NAG 

Group 

Experiment 

Day 

Ug/ml 

ue/ml 

U/L 

U/L 

Saline 

Cl 

1 

240.0 

52.5 

43.1 

1.7 




73.9 

15.2 

5.3 

0.2 

WTC8 

Cl 

1 

191.9 

40.5 

46.1 

2.7 




9.5 

3.1 

3.6 

0.3 

WTC13 

Cl 

1 

201.8 

40.6 

40.6 

2.9 




16.3 

4.5 

4.4 

0.2 

WTCF 

Cl 

1 

200.4 

42.9 

44.6 

1.7 




11.9 

3.4 

3.3 

0.2 

NIST 

Cl 

1 

257.1 

52.6 

56.8 

4.5 




16.3 

4.1 

5.0 

0.4 

Saline 

Cl 

3 

274.6 

55.9 

37.5 

3.1 




65.4 

10.0 

11.1 

1.3 

WTC8 

Cl 

3 

156.1 

34.9 

35.2 

2.3 




13.3 

4.6 

1.8 

0.1 

WTC13 

Cl 

3 

203.8 

47.1 

36.0 

2.7 




26.4 

8.1 

2.2 

0.2 

WTCF 

Cl 

3 

197.5 

45.4 

37.2 

2.9 




15.9 

4.5 

2.8 

0.4 

NIST 

Cl 

3 

220.4 

45.2 

40.2 

4.3 




17.3 

4.6 

5.2 

0.6 

Saline 

C2 

1 

307.8 

75.5 

44.5 

2.9 




95.7 

28.0 

18.9 

0.2 

WTCll 

C2 

1 

202.0 

44.8 

48.1 

2.4 




30.8 

7.7 

6.2 

0.7 

WTCB 

C2 

1 

242.5 

45.2 

55.2 

2.9 




56.4 

6.6 

13.0 

0.8 

WTCC 

C2 

1 

194.8 

42.2 

34.6 

1.7 




13.5 

4.6 

2.6 

0.1 

WTCE 

C2 

1 

194.5 

43.5 

39.5 

3.0 




11.7 

3.6 

2.7 

0.4 

Saline 

C2 

3 

180.8 

46.8 

29.5 

2.3 




21.1 

6.1 

4.9 

0.3 

WTCll 

C2 

3 

213.0 

49.2 

50.1 

4.4 




64.7 

11.5 

15.0 

2.3 

WTCB 

C2 

3 

131.8 

32.5 

32.4 

2.2 




12.2 

4.6 

3.6 

0.1 

WTCC 

C2 

3 

167.2 

44.8 

30.0 

2.9 




16.4 

5.8 

1.7 

0.2 

WTCE 

C2 

3 

190.2 

51.0 

29.4 

2.5 




23.4 

8.1 

2.1 

0.2 


“ Values shown are means (in bold) and SEM immediately below means (n=8 per group, except Saline sub-experiment 
C2: n=4). Significant differences shown are within sub-experiments only. Heavy solid-line boxes; NIST significantly 
different from WTC8. Significant overall Day effects were found for Protein (sub-experiment C2), and LDH (both 
sub-experiments). 


39 





























400-1 


£ 300- 
'w) 


.S 200- 

"S 

O 

100 - 


Protein 


H Saline (Cl) 


DD 

H 


£ 

3 

Xi 


100 - 

80- 

60- 

40- 

20 - 

0 




Day 1 


1 




dznn WTC8 
WTC13 



Day 3 


Albumin 


X 


] Saline (Cl) 
] WTC8 
WTC13 
WTCF 
NIST 


X, 



Day 1 


Day 3 



80 n 


LDH 


Day 1 


10-1 

8 - 

3 6 - 


O 

< 

z. 


i- . J Saline (Cl) 

3 WTC8 
WTC13 



Day 3 


NAG 


□ 




Saline (Cl) 
WTC8 
WTC13 
WTCF 
■■■NIST 



Day 1 


Day 3 




Figure 17. Experiment C: BAL supernatant biochemistry. See next page for figure legend. 


40 
























































































































































































































































































































































































































































Figure 17. (previous page.) Experiment C: Bronchoalveolar 
lavage supernatant proteins and enzymes recovered from mice 
exposed to saline vehicle, NIST 1649a, or WTC PM samples 
from individual collection sites and tested 1 or 3 days later (n 
= 8 per group except Saline sub-experiment C2: n = 4). “ 
Significantly greater protein values in NIST group vs. WTC8 
group. Other significant differences were found due to Day 
of sacrifice or interactions between Day and Treatment, but 
there were no other effects due to Treatment alone. 


6. Summary. Examination of the effects of WTC 
PM collected from different locations surrounding the 
WTC site showed that all samples were capable of 
inducing pulmonary inflammation and 
hyperresponsiveness to Mch aerosol, although overt lung 
damage as determined by biochemical parameters of lung 
injury was minimal. The neutrophilic response was 
substantially greater for all individual site WTC PM 


samples compared with the response induced by the 
pooled WTCX sample in Experiment A, although differing 
responsiveness of different shipments of mice could 
account for this finding, and a direct comparison would be 
necessary to determine if there is a difference. Numbers 
of neutrophils declined from Day 1 to Day 3 after 
oropharyngeal aspiration, as determined by both BAL and 
histopathologic examination. Other cell types appeared to 
be more persistent or increase from Day 1 to Day 3 
(especially lymphocytes in sub-experiment C1), but these 
were not large changes. Respiratory responsiveness to 
Mch aerosol was significantly increased in all WTC 
groups compared with saline controls, although mice 
exposed to WTC 13 were less responsive than other WTC 
groups. The degree of Mch hyperresponsiveness in the 
WTC groups of Experiment C appeared to be comparable 
to that from the WTCX group in Experiment A. 

No particular geographical significance could be 
deduced from the patterns of responses induced by the 
individual WTC PM samples. The one group which had 


Table 21. Experiment C - Summary of Treatment-Related Histopathologic Findings in Mice 1 or 3 Days after 


Intratracheal Instillation of Particulate Matter Samples “ 


Treatment 

Group 

Sub- 

Experiment 

Day 

Bronchiole, 

Inflammation, 

Subacute, Focal 

Bronchiole, 

Pigment, 

Macrophage, Focal 

Bronchiole, 

Pigment, 

Free, Focal 

Incidence 

Severitv 

Incidence Severitv 

Incidence 

Severitv 

WTC 13 

Cl 

1 

8/8 

2.1 

8/8 

2.0 

4/8 

0.6 

WTCE 

C2 

1 

8/8 

2.0 

8/8 

1.9 

2/8 

0.3 

NIST 

Cl 

1 

8/8 

1.9 

7/8 

2.0 

7/8 

0.9 

WTC 11 

C2 

1 

8/8 

1.3 

7/8 

0.9 

0/8 

0.0 

WTCF 

Cl 

1 

8/8 

1.3 

6/8 

0.8 

0/8 

0.0 

WTC8 

Cl 

1 

6/8 

1.1 

6/8 

0.8 

0/8 

0.0 

WTCB 

C2 

1 

6/8 

1.1 

6/8 

0.8 

0/8 

0.0 

WTCC 

C2 

1 

6/8 

0.8 

4/8 

0.5 

0/8 

0.0 

Saline 

Cl 

1 

1/8 

0.1 

0/8 

0.0 

0/8 

0.0 

NIST 

Cl 

3 

8/8 

2.1 

8/8 

2.0 

0/8 

0.0 

WTCl 1 

C2 

3 

6/8 

1.1 

2/8 

0.3 

0/8 

0.0 

WTCE 

C2 

3 

6/8 

0.8 

6/8 

0.8 

0/8 

0.0 

WTC8 

Cl 

3 

4/8 

0.8 

1/8 

0.1 

0/8 

0.0 

WTC 13 

Cl 

3 

4/8 

0.6 

3/8 

0.4 

0/8 

0.0 

WTCB 

C2 

3 

3/8 

0.4 

2/8 

0.3 

0/8 

0.0 

WTCF 

Cl 

3 

3/8 

0.4 

1/8 

0.1 

0/8 

0.0 

WTCC 

C2 

3 

2/8 

0.3 

1/8 

0.1 

0/8 

0.0 

Saline 

Cl 

3 

0/7 

0.0 

0/7 

0.0 

0/7 

0.0 


Saline-instilled control mice in sub-experiment C2 were not examined. Incidence denotes number of mice in group with 
finding / total number of mice examined. Average severity score for the group is shown based on the following scoring 
system: 0 = not present, 1 = minimal, 2 = slight/mild, 3 = moderate, 4 = moderately severe, 5 = severe/high. Groups 
are arranged in descending order of severity within each post-exposure day, first by severity of focal subacute 
bronchioiar inflammation, and then by severity of focal bronchiolar pigmented macrophages. 


41 























Figure 18. Experiment C. Representative micrographs of lesions occuring in lungs of mice 1 or 
3 days after intratracheal instillation of 100 pg PM sample or saline vehicle (all panels same 
magnification: bar length = 100 pm). A. Saline-instilled control mouse (#301), Day 1, with no 
remarkable findings. B. Mouse #349 instilled with WTCC, Day 1, showing minimal degree of focal 
subacute bronchiolar inflammation (FSBI). C. Mouse #291 instilled with NIST, Day 1, with 
moderate degree of FSBI. D. Mouse #270 instilled with WTC13, Day 1, with moderate degree of 
FSBI. E. Mouse #300 instilled with NIST, Day 3, with slight/mild degree of FSBI. F. Mouse #280 
instilled with WTC13, Day 3, with minimal degree of FSBI. 


lower Mch responsiveness (WTC13) was centrally located 
only 0.1 mile southeast of the center of Ground Zero. The 
WTCF sample was blown into a building at 120 Broadway 
and collected on an undisturbed marble staircase. The 
responses caused by this “indoor” sample were quite 
similar to those caused by the other “outdoor” WTC PM 
samples. 


In general, responsiveness to Mch aerosol and 
pulmonary inflammation were not well correlated. Mice 
in the WTC 13 group had one of the largest neutrophilic 
and eosinophilic responses, yet had a significantly lower 
degree of Mch responsiveness. Mice in the WTCC group 
had perhaps the greatest response to Mch challenge (not 
significantly different from WTCB, WTCE, or WTCl 1), 


42 





yet their neutrophil and eosinophil responses were low 
relative to the other WTC groups. As noted previously, a 
lack of correlation between inflammation and airway 


hyperresponsiveness is not uncommon (Alvarez et al., 
2000; Smith and McFadden Jr., 1995). 


43 






IV. Discussion 


Samples of fallen dust were collected at various 
locations in the immediate vicinity of the WTC site one 
and two days after the WTC disaster, and were examined 
by several physical and chemical techniques. Both coarse 
unfractionated and fine size-fractionated WTC PM 
samples were composed primarily of calcium-based 
compounds such as calcium sulfate (gypsum) and calcium 
carbonate (calcite; the main component of limestone). 
These and other compounds and elements found in the 
WTC PM samples are indicative of crustal material- 
derived building materials such as cement, concrete 
aggregate, ceiling tiles, and wallboard. Both gypsum and 
calcite irritate the mucus membranes of the eyes, nose, 
throat, and upper airways (Stellman, 1998). Calcium 
carbonate dust causes coughing, sneezing, and nasal 
irritation (NLM, 2002). These minerals are often 
contaminated with small amounts of silica, which is the 
main concern for occupational health hazards (Stellman, 
1998). Minor amounts of silica (quartz) were detected in 
the WTC PM samples 

Our chemical analysis generally agrees with the 
extensive analysis of WTC PM performed by the USGS 
(USGS, 2002). Levels of carbon were relatively low, 
suggesting that combustion-derived particles did not form 
a significant fraction of these samples recovered in the 
immediate aftermath of the destruction of the towers. 
Lastly, there was no evidence of significant asbestos 
contamination of the samples used in these studies, 
although the physical analyses conducted were not 
specifically focused on definitive asbestos quantitation. 
As of May 23,2002, the U.S. EPA had analyzed 9,544 air 
samples in Lower Manhattan since September 11, and 
found elevated levels of asbestos in only 21 samples (EPA, 
2002 c). 

The effects of exposure to samples of WTC PM, 5 on 
respiratory parameters, pulmonary inflammation, and lung 
injury were investigated in young adult female CD-I mice, 
an outbred strain expected to have significant variability 
in biological responses, in three separate experiments. A 
pooled sample of WTC PM 2 5 composed of roughly 
equivalent amounts of samples from 7 different locations 


around the WTC site caused a mild degree of pulmonary 
inflammation in mice (7% neutrophils in BAL fluid), and 
had no effect on parameters of acute lung injury at a dose 
of 100 pg instilled directly into the lungs. ROFA, a toxic 
positive control fine PM sample, caused a much higher 
degree of lung inflammation and lung injury at the same 
dose. However, mice instilled with 100 pg pooled WTC 
PM 25 had highly significant increases in airway 
responsiveness to methacholine (Mch) aerosol challenge, 
which were significantly greater than that of ROFA. Mice 
exposed to lower doses of pooled WTC PM 2 5 (10 pg and 
31.6 pg) and mice exposed by nose-only inhalation 
(estimated to have about 14 pg WTC PM 2 5 deposited in 
the respiratory tract) did not have any biologically 
significant changes in methacholine responsiveness or 
neutrophilic inflammation. These dose-response 
relationships and the lack of effect in nose-only exposure 
suggest that inhalation of relatively high doses of WTC 
PM 2 5 are necessary to elicit respiratory effects in people. 

Mice exposed to samples of WTC PM 2 5 from the 7 
individual sites around Ground Zero had greater lung 
inflammation (2 to 4-fold) than mice exposed to the WTC 
PM 25 sample pooled from these sites. These findings 
occurred in separate experiments and would need to be 
confirmed by a direct comparison, but nonetheless all 
groups of mice exposed to the individual site samples 
developed hyperresponsiveness to Mch aerosol challenge, 
similar to mice exposed to the pooled sample. No 
particular pattern of responses was found corresponding to 
the geographical location where the samples were taken. 
Pulmonary inflammation in mice exposed to individual 
site WTC PM 2 5 samples diminished from 1 day to 3 days 
after exposure, although hyperresponsiveness to Mch 
aerosol did not diminish significantly. Further 
experiments would be necessary to determine the 
persistence of pulmonary responses in mice, which may 
lead to insights into whether any WTC PM-associated 
effects which may exist in people are persistent. 

The results of these studies should be examined in the 
context of previous studies of the effects of 


44 









environmentally relevant PM samples in rodents. Rats 
were intratracheally instilled with 2.5 mg (~ 8.3 mg/kg) of 
various emission source and urban ambient air PM 
samples (Costa and Dreher, 1997), a dose about twice as 
high, based on body weight, as the 100 pg WTC PM 25 
dose in mice (~4 mg/kg). Oil fly ashes and urban ambient 
air PM samples (including a ROFA similar to the one used 
in the present study and NIST 1649a) induced strong 
neutrophilic responses 24 hr after exposure, while 
biochemical markers of lung injury were lower in the 
urban air PM samples compared with the oil fly ash 
samples. ROFA at this dose induced airway 
hyperresponsiveness in rats which persisted at least 4 days, 
and was greater than that observed in an urban ambient air 
PM sample (Pritchard et al., 1996). The fact that WTC 
PM 2 5 induced a significantly greater degree of airway 
hyperresponsiveness in mice than ROFA, which is used as 
a toxic positive control particle in many studies, suggests 
a very significant respiratory effect of a relatively high 
dose exposure to WTC PM 2 5. 

Some people were exposed acutely to high 
concentrations of dust in the WTC disaster, and 
subsequently developed wheezing or symptoms of sensory 
irritation, such as cough and irritation of the nose and 
throat. These effects resemble, in some respects, the 
reactive airways dysfunction syndrome (RADS). RADS 
can occur after single or multiple high-level occupational 
exposures to an irritating vapor, fume, or smoke (Gautrin 
et al., 1999). Effects can occur within minutes or hours 
after exposure, and include cough, dyspnea, and wheezing. 
Clinical tests can show airways obstruction, persistent 
airway hyperresponsiveness, and inflammation. The 
recovery process appears to be dependent on the initial 
degree of injury. The effects of a high dose exposure to 
WTC PM 2 5 in mice (100 pg) appear to mimic at least 
some of these responses, especially the significant increase 
in airway hyperresponsiveness to Mch. It is important to 
note that WTC PM 2 5 -induced pulmonary inflammation, 
although significantly greater than in control mice, was not 
as robust as one might expect in a realistic animal model 
of RADS. However, the degree to which inflammation 
and airway hyperresponsiveness are associated in RADS 
is not clear (Gautrin et al., 1999). Examination of other 
time points would be necessary to determine the 
persistence of WTC PM-induced airway 
hyperresponsiveness in mice and its similarity to RADS. 

Close examination of the data suggested that 
individual mice within the outbred CD-I strain vary in 
sensitivity to the effects of WTC PM 25 . Certain 
individuals within the human population may also have 
particular susceptibility to the hazards posed by exposure 


to WTC PM 25 . It is known that some asthmatic 
individuals are hyperresponsive to nonspecific irritants 
such as cold dry air (Anderson and Daviskas, 2000) or 
cigarette smoke (Bonham et al., 2001). This 
subpopulation is likely to be at high risk for development 
of dust-induced airways obstruction (Donaldson et al., 
2000; Peden, 2001; Nel et al., 2001). Very few studies 
have been published regarding the effects of alkaline 
aerosols on pulmonary function in asthma. One study 
reported that inhalation of high concentrations of an 
alkaline aerosol (pH 9.8 to 10.3) had no significant effect 
on irritant symptoms or specific airways resistance in mild 
asthmatic patients (Eschenbacher, 1991). However, this 
aerosol was composed of a simple mixture of sodium 
carbonate, sodium bicarbonate, and sodium hydroxide. 
The chemical composition of the alkaline (pH 8.88 to 
10.00) WTC PM 2 5 is much more complex and interactions 
of numerous chemical species may be associated with 
development of airway hyperresponsiveness to 
methacholine or other bronchoconstrictors. 

How does the dose of 100 pg WTC PM 2 . 5 , which 
caused bronchiolar inflammation and airway 
hyperresponsiveness in mice, relate to exposure of people 
at the WTC site? Because inflammation was observed 
mainly in the airways, and airway hyperresponsiveness is 
mainly due to dysfunction of airway smooth muscle 
(Fredberg, 2000), the dose metric which is probably most 
relevant is dose per surface area of the tracheobronchial 
(TB) region of the respiratory tract. The TB region is 
defined as the airways (excluding the nasal (head) region) 
from the trachea down to the terminal bronchioles 
(Overton et al., 2001). Therefore, to assess the risks of 
exposure in people, the concentrations of WTC PM 2 5 in 
air which could produce doses per TB surface area in 
humans equivalent to that in mice should be calculated. 
These WTC PM 2 5 concentrations may be estimated (Table 
22) using the following assumptions: 1) The mouse 
alveolar pulmonary surface area can be estimated from an 
allometric equation based on body weight (Jones and 
Longworth, 1992), and the TB surface area is very small 
in comparison to the alveolar surface area (Overton et al., 
2001); 2) Oropharyngeal aspiration bypasses the mouse 
nose and spreads the dose of WTC PM 2 5 evenly over the 
TB and pulmonary alveolar surface areas of the mouse 
lung; 3) The human TB dose per surface area, selected to 
match the mouse dose per surface area, does not clear from 
the lung in the time frame of exposure to WTC PM 2 5 (an 
8 -hour work shift was selected); and 4) The model of the 
fraction of inhaled PM 2 5 (model particles with MM AD = 
1, CTg = 2.5, and density = 1 g/cc) deposited in the TB 
region (Freijer et al., 1999) assumes a reference 30 year- 


45 







Table 22 Estimation of WTC PMj j Concentrations Required to Produce Human Doses Equivalent to Mouse 
Doses Used in WTC2001 Study 


Dose deposited in mouse tracheobronchial and pulmonary regions (pg) 

10 

31.6 

100 

Mouse alveolar pulmonary surface area (m^) ® 

0.103 

0.103 

0.103 

Mouse dose per tracheobronchial (TB) or pulmonary surface area (mg/m^) ’’ 

0.097 

0.307 

0.973 

Human TB surface area (m^)' 

0.415 

0.415 

0.415 

Total human TB dose equivalent to mouse TB dose (mg/m‘ x m") 

0.040 

0.128 

0.404 

Deposition fraction in human TB region' 

0.066 

0.066 

0.066 

Total inhaled dose in mg (total human TB dose / TB deposition fraction) 

0.612 

1.932 

6.115 

Quantity of air breathed in 8 hr workshift at ventilation of 30 L/min (m’) 

14.4 

14.4 

14.4 

WTC PMj 5 concentrations required to produce human doses equivalent to 
mouse doses used in WTC2001 Study (pg/m^) 

42 

134 

425 


® From Jones and Longworth (1992) calculated allometric equation: Mammalian alveolar pulmonary surface 
area in m^ = 3.36 x (Wt, where weight = 0.024 kg (average mouse weight in all studies). 

Tracheobronchial surface area is minimal in comparison to alveolar surface area and can be ignored in 
calculation. 

Assumes dose is spread out evenly over tracheobronchial and pulmonary alveolar regions. 

' Based on 30 year old, 5' 10" male with functional residual capacity (FRC) of 3300 ml (Overton et al., 

2001 ). 

Calculations assume no clearance of particles after deposition in human respiratory tract. 

® Calculations made with Multiple Path Particle Deposition model version 1.11 (Freijer et al., 1999) which 
assume human Yeh-Schum 5-lobe model, FRC = 3300 ml (appropriate for 30 year old, 5'10" male), upper 
respiratory tract volume = 50 ml, density of particles = 1 gm / cc, diameter = 1 |j,m MMAD, inhalability 
adjustment on, og = 2.5, breathing frequency = 15 min'’, tidal volume = 2000 ml, minute volume = 30 
L/min, inspiratoryiexpiratory ratio = 1, and oronasal mouth breathing. 

^ Estimate of minute ventilation during moderate to heavy sustained work (Astrand and Rodahl, 1986). 


old 5' 10" male breathing oronasally with a minute 
ventilation of 30 L/min (estimate during moderate to heavy 
sustained work; Astrand and Rodahl, 1986). The total 
human TB dose and the fraction deposited in the TB 
region are used to back-calculate the total inhaled dose of 
PM 2 5 . The total inhaled dose divided by the quantity of 
air breathed in a typical 8 -hour work shift yields the 
concentration of PMj 5 in the WTC work or neighborhood 
environment required to produce human doses equivalent 
to the mouse doses used in the WTC2001 study (Table 
22). These calculations show that under these conditions, 
concentrations of 42, 134, and 425 pg/m^ WTC PM 2 5 
would produce human doses per TB surface area 
equivalent to the mouse doses of 10 , 31.6, and 100 pg, 
respectively. Obviously many factors may cause wide 
variations in the calculation of dose, and extrapolation of 
responses from the mouse to the human involves another 
dimension of uncertainty which was not considered, but it 
seems reasonable to say that a healthy worker breathing 
heavily in the dusty environment generated after the 
collapse of the towers could have inhaled enough PM 2 5 to 


approximate the 100 pg dose in the mouse. Therefore, 
inhalation of a very high concentration of WTC PM 2 5 (e.g. 
-425 pg/m^) over a short period of time (8 hr) could have 
contributed to development of pulmonary inflammation, 
airway hyperresponsiveness, and manifestations of sensory 
irritation such as cough. Individuals who are especially 
sensitive to inhalation of dusts, such as asthmatics, may 
experience these effects at lower doses of inhaled WTC 
PM 2 5 . However, most healthy people would not be 
expected to respond to moderately high WTC PM 2 5 levels 
(130 pg/m^ or less for 8 hours) with any adverse 
respiratory responses. The effects of chronic or repeated 
exposures to lower levels of WTC PM 2 5 , or the persistence 
of any respiratory effects are unknown and were not 
components of this study. The persistence of any effects 
of inhaled WTC PM 2 5 , if similar to RADS, would be 
expected to depend on the dose initially deposited in the 
respiratory tract. 

It is important to consider several limitations of these 
studies. First, most of the experiments used oropharyngeal 
aspiration to deliver PM samples to the respiratory tract 


46 










rather than more physiologically relevant inhalation 
exposure methodology. We believe that utilizing 
oropharyngeal aspiration, as described in the Experimental 
Design section, had many advantages and was necessary 
in these circumstances. However, this report indicates that 
future studies may be needed to more closely examine 
bronchoconstriction and sensory irritation during 
inhalation exposure to WTC PM in mice and in guinea 
pigs, a species known to be especially sensitive to sensory 
irritants (Costa and Schelegle, 1999). Secondly, these 
studies only evaluated short-term toxicological effects 
(endpoints were examined 1, 3, or 6 days after exposure) 
after acute exposure and no direct information is provided 
on the long term effects of acute or chronic exposures to 
WTC PM 2 5 . Thirdly, gaseous and vapor-phase toxicants 
(e.g. dioxin and volatile organic compounds such as 
benzene) were certainly released, especially during the 
fires which continued for months after September 11 
(EPA, 2002c). The collection and processing techniques 
described in this report do not allow investigation of these 
important toxic species, nor are the interactions of 
particles with gases or organic vapors considered (Mautz 
et al., 2001). Finally, these studies only examined fine 
PMj 5, while the toxicity of coarse mode and larger size 
PM fractions were not investigated. However, it is 
important to remember that the size-fractionation 


techniques employed in this report are not absolute, and 
significant quantities of PM > 2.5 pm are present in the 
samples. Furthermore, analysis of the WTC PM 2 5 and 
PM 53 samples showed that they were similar in 
composition (Tables 3 and 5), suggesting that only 
differences in respiratory tract deposition patterns of fine 
and coarse WTC PM would affect biological responses. 
Coarse mode PM may be more relevant for upper airways 
sensory irritation because larger particles will mainly 
deposit in the upper airways where sensory innervations 
are predominant (Costa and Schelegle, 1999). However, 
chronic effects of fine PM may be greater than coarse PM 
since it can be inhaled more deeply and deposit in 
peripheral regions of the lungs, and is more slowly 
cleared. Coarse PM is much less inhalable in small 
rodents than in humans, and less is deposited in the 
respiratory tract (Menache et al., 1995). Consequently, 
interpretation of results derived from exposure of mice to 
coarse PM is problematic, and small rodents are probably 
not the ideal species to study effects of coarse PM. 
Nevertheless, because upper airways irritant responses 
seem to be so important in people exposed to WTC- 
derived dust, future studies should examine the specific 
toxicity of coarse WTC PM on respiratory responses in 
appropriate animal models. 


47 






V. Quality Assurance Statement 


U.S. EPA World Trade Center Research Project: 

“Toxicological Effects of Fine Particulate Matter Derived 
from the Destruction of the World Trade Center” 


Page 1 of2 

The study “T oxicological Effects of Fine Particulate Matter Derived from the Destruction of the World T rade Center” 
was conducted by the Pulmonary Toxicology Branch, Experimental Toxicology Division (ETD), National Health and 
Environmental Effects Research Laboratory (NHEERL), Office of Research and Development, U.S. Environmental 
Protection Agency, Research Triangle Park, NC, in compliance with NHEERL QA Guidelines. Results of these 
inspections were reported directly to the Principal Investigator (PI) of the Study, Dr. Stephen Gavett. Critical phases in 
the study were audited. 


Date of Inspection 

October 29, 2001 
October 31, 2001 
November 2, 2001 
November 2, 2001 
November 8, 2001 
November 5-14,2001 

November 15, 2001 
November 19, 2001 
November 26, 2001 
November 27,2001 

December 3, 2001 


Item Inspected 

Particulate Matter (PM) filters delivered to EPA. 

Attempted scraping of PM from filters. 

Approval of study protocol. 

Extraction of PM from filters. 

Shipment of WTC dust samples for endotoxin testing. 

Conduct of Experiment A1 -A3: weighing of samples and mice, randomization of mice, 
dosing of mice, BUXCO, DLCO, BAL, cell counts, methacholine responses, lung 
samples. 

Delivery of NIST samples and blank filters. 

Delivery of #3 Cortland Sample. Shipment of NIST samples for endotoxin testing. 
Delivery of Experiment B inhalation sample (WTC 3). 

Conduct of Experiment B (Day 0): placing and removal of mice in inhalation chambers, 
operation of inhalation pump. Shipment of #3 Cortland Sample Back to NYU. 

Completion of Experiment B (Day 6): Nasal fixation. 

Receipt of endotoxin results on WTC dust samples. 


48 




Quality Assurance Statement 


Page 2 of 2 


Date of Inspection 

December 4, 2001 
December 11, 2001 

December 27, 2001 
January 15-17,2002 

January 16, 2002 

January 16-25, 2002 
January 30, 2002 
March 15, 2002 
March 4-19, 2002 


Item Inspected 

XRP/XRD laboratory tour. Shipment of 96 mice heads to Michigan State University. 

Conduct of Experiment C (Day 0): Dosing of Mice. 

XRF/XRJD technical meeting. Receipt of NIST endotoxin test results. 

Shipment of 184 lung tissues for histopathological analysis. 

Technical Systems Review of project. Interviews with study personnel and inspection 
of project data and records. 

Shipment of six PM samples, and 12 PM samples and 10 filter samples for chemical 
analysis. 

Data audit of spreadsheets against notebooks. 

Shipment of 10 liquid samples for chemical analysis. 

Transfer of custody of 12 PM Samples from the EPA Chemist to the PI. 

Data audit of Draft Final Report. 


The Quality Assurance Manager of ETD and the Director of Quality Assurance for NHEERL have determined by 
the above review process that the conduct of this project was in compliance with EPA quality requirements and the 
operating procedures and study protocol (Intramural Research Protocol No.: IRP-NHEERL-H/ETD/PTB/SHG/01-01- 
000). Furthermore, the results accurately reflect the raw data obtained during the course of the study. 


/s/ _ 03/22/2002 

Thomas J. Hughes, ETD QA Manager Date 

/s/ _ 03/22/2002 

Brenda T. Culpepper, NHEERL Director of QA Date 


49 


















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53 












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